Facing and Quality Control in Microtomy

ABSTRACT

The present disclosure also relates to systems and methods for quality control in histology systems. In some embodiments, a method is provided that includes receiving a tissue block comprising a tissue sample embedded in an embedding material, imaging the tissue block to create a first imaging data of the tissue sample in a tissue section on the tissue block, removing the tissue section from the tissue block, the tissue section comprising a part of the tissue sample, imaging the tissue section to create a second imaging data of the tissue sample in the tissue section, and comparing the first imaging data to the second imaging data to confirm correspondence in the tissue sample in the first imaging data and the second imaging data based on one or more quality control parameters.

RELATED APPLICATIONS

This application is a continuation patent application of U.S. patentapplication Ser. No. 17/182,139, filed Feb. 22, 2021, which claims thebenefit of and priority to U.S. Provisional Application Ser. No.62/980,201, filed on Feb. 22, 2020, U.S. Provisional Application Ser.No. 62/980,203, filed on Feb. 22, 2020, U.S. Provisional ApplicationSer. No. 62/980,202, filed on Feb. 22, 2020, U.S. ProvisionalApplication Ser. No. 62/980,194 filed on Feb. 22, 2020, and U.S.Provisional Application Ser. No. 63/134,399, filed on Jan. 6, 2021, eachof which are incorporated herein by reference in their entireties.

FIELD

The present invention relates to quality control of cut tissue sectionstransferred from a biological tissue sample block to a slide, includingfacing, tracking, and mechanical quality control. In particular, thiscan be achieved using an automated system.

BACKGROUND

Traditional microtomy, the production of postage-stamp sized,micron-thin tissue sections for microscope viewing, is a delicate, timeconsuming manual task. In the process, a microtome cuts a tissue blockconsisting of tissue sample, enclosed in a supporting block of embeddingmaterial such as paraffin wax. The microtome holds a blade aligned forcutting slices from one face of tissue block—the block cutting face. Acommon type, the rotary microtome, linearly oscillates a chuck holdingthe block with the cutting face in the blade-cutting plane. Combinedwith incremental advancement of the block cutting face into the cuttingplane, the microtome successively shaves thin tissue sections off theblock cutting face. For sections with paraffin wax embedding medium, anoperator carefully picks up these tissue sections and floats them onwarm water. The water gently de-wrinkles and reduces deformation fromcutting. Finally, an operator moves the sections from water ontomicroscope slides for further processing.

Recent advancements in the digital imaging of tissue sample sectionshave made it desirable to slice blocks of specimen very quickly. By wayof example, where tissues are sectioned as part of clinical care, timeis an important variable in improving patient care. Every minute thatcan be saved during sectioning of tissue for intra-operativeapplications of anatomic pathology, for example in examining margins oflung cancers to determine whether enough tissue has been removed, is ofclinical value. To create a large number of sample sections quickly, itis desirable to automate the process of cutting tissue sections from aspecimen block by a microtome blade and facilitating the transfer of cuttissue sections to an adhesive tape or other transfer medium withoutreducing section quality. Additionally, the large number of tissuesample sections cut from the block need to be transferred to microscopeslides for evaluation.

Quality control for tissue samples is important. Inadequate qualitycontrol can adversely affect pathology, leading to inaccurate assessmentof tissue. However, currently quality control of tissue sectionsdeposited on glass slides is a resource consuming task.

This comparison is done in manual transfer of tissue to the slides,however, it is often difficult to differentiate the tissue from theparaffin, leading to inaccurate comparisons and thus inadequate qualitycontrol. Further, inability to properly assess the tissue results inlack of knowledge that an insufficient tissue section is placed on theslide. It also inhibits the assessment of whether the tissue section onthe slide has been damaged.

The present disclosure overcomes the problems and deficiencies of thecurrent workflow by the implementation of methods and systems thateliminate or at least significantly decrease the quality control issuesand the risk of the mismatch between the slide label and the tissuesection.

SUMMARY

The present disclosure relates to systems and methods for facing atissue block. In some embodiments, a method is provided for facing atissue block that includes imaging a tissue block to generate imagingdata of the tissue block, the tissue block comprising a tissue sampleembedded in an embedding material, estimating, based on the imagingdata, a depth profile of the tissue block, wherein the depth profilecomprises a thickness of the embedding material to be removed to exposethe tissue sample to a pre-determined criteria, and removing thethickness of the embedding material to expose the tissue to thepre-determined criteria.

In some embodiments, the method can further include progressivelyremoving one or more sections from a tissue block comprising a tissuesample embedded in an embedding material, imaging the one or moresections to generate imaging data associated with the one or moresections, and confirming, based on the imaging data, that the tissuesample is exposed to the predetermined criteria. In some embodiments,the tissue block is imaged with a structured light to determine thedepth profile.

In some embodiments, a method for facing a tissue block is provided andcan include progressively removing one or more sections from a tissueblock comprising a tissue sample embedded in an embedding material,imaging the one or more sections removed from the tissue block togenerate imaging data associated with the one or more sections, anddetermining, based on the imaging data, when a sufficient number of theone or more sections have been removed from the tissue block to exposethe tissue sample to a predetermined criteria.

In some embodiments, the method can further include imaging the tissueblock, prior to removing the one or more sections, to generate abaseline imaging data of the tissue sample. In some embodiments, themethod can further include determining an expected outline, size, orshape of the tissue sample from the baseline imaging data. In someembodiments, the method can further include determining a depth profileof the embedding material from the baseline imaging data to remove asufficient amount of the embedding material to expose the tissue sampleto the predetermined criteria, and illuminating the tissue block with astructured light in a UV range.

In some embodiments, the method can further include comparing theimaging data of the sections including a tissue sample with the baselineimaging data to determine when the tissue sample has been sufficientlyexposed. In some embodiments, the imaging data of the sections comprisesimaging data of the one or more sections on the tissue block, on atransfer medium, or on a slide. In some embodiments, an outline, size,or shape of the tissue sample in the one or more sections is compared toan outline, size, or shape of the tissue sample expected from thebaseline imaging data. In some embodiments, the method can furtherinclude determining a depth profile by one or more of disparity, fromfocus, or light field imaging, and increasing contrast between thetissue sample and the embedding material.

In some embodiments, a method can be provided for facing a tissue blockthat includes removing, from a tissue block comprising a tissue sampleembedded in an embedding material, a thickness of the embedding materialconfigured to expose the tissue sample to a predetermined criteria,subsequently to removing the thickness, progressively removing one ormore sections from the tissue block, imaging the one or more sectionsremoved from the tissue block to generate imaging data associated withthe one or more sections, and confirming, from imaging data, that thetissue sample has been exposed to the predetermined criteria.

In some embodiments, a histology system can be provided that includes amicrotome configured to progressively remove one or more sections from atissue block, the tissue block comprising a tissue sample embedded in anembedding material, and a vision system associated with the microtome.The vision system can include an illumination system configured toilluminate the tissue block comprising a tissue sample embedded in anembedding material, an imaging system configured to image the tissueblock to generate imaging data associated with the tissue block, and aprocessor in communication with the vision system, the processor beingprogramed to receive the imaging data and determine, based on theimaging data, when the tissue block has been sufficiently faced by themicrotome.

In some embodiments, the processor is further programed to determinewhen the tissue block has been sufficiently faced by recognizing anamount of exposed tissue sample. In some embodiments, the processor isfurther programmed to determine an expected outline, size, or shape ofthe tissue sample from a baseline imaging data generated by imaging thetissue block with structured light prior to removing the one or moresections from the tissue block. In some embodiments, the illuminationsystem is configured to illuminate the tissue block with structuredlight.

In some embodiments, the histology system can further include a transfermedium to transfer one or more sections comprising a tissue sample fromthe tissue block to one or more slides and wherein to processor isfurther programmed to compare the one or more sections on the tissueblock, on the transfer medium, or on the one or more slides to abaseline imaging data generated by imaging the tissue block with UVlight prior to removing the one or more sections from the tissue block.

In some embodiments, a vision system is provided that includes anillumination system configured to illuminate a tissue block comprising atissue sample embedded in an embedding material, an imaging systemconfigured to image the tissue block to generate imaging data of thetissue block, and a processor in communication with the imaging system,the processor being programed to receive the imaging data and determine,based on the imaging data, an exposure of the tissue sample to apredetermined criteria.

The present disclosure also relates to systems and methods for qualitycontrol in histology systems. In some embodiments, a method is providedthat includes receiving a tissue block comprising a tissue sampleembedded in an embedding material, imaging the tissue block to create afirst imaging data of the tissue sample in a tissue section on thetissue block, removing the tissue section from the tissue block, thetissue section comprising a part of the tissue sample, imaging thetissue section to create a second imaging data of the tissue sample inthe tissue section, and comparing the first imaging data to the secondimaging data to confirm correspondence in the tissue sample in the firstimaging data and the second imaging data based on one or more qualitycontrol parameters.

In some embodiments, the tissue section is non-conforming if there is nocorrespondence in one or more quality control parameters in the tissuesample in the first imaging data and the second imaging data. In someembodiments, the one or more quality control parameters include one ormore of shape of the tissue sample, size of the tissue sample, or one ormore mechanical damages. In some embodiments, the method can furtherinclude transferring, using a transfer medium, the tissue section to aslide, and the second imaging data comprises an imaging data of thetissue section on the transfer medium or an imaging data of the tissuesection on the slide. In some embodiments, the method can furtherinclude comparing at least two of the first imaging data, the imagingdata of the tissue section on the transfer medium or the imaging data ofthe tissue section on the slide.

In some embodiments, the tissue section is non-conforming if there is nocorrespondence in the shape or the size of the tissue sample in thefirst imaging data and the second imaging data. In some embodiments, theone or more mechanical damages are selected from the group consisting oftearing, shredding, blade marks, wrinkling, cracking, bubbles,insufficient tissue sample, incomplete tissue sample. In someembodiments, the method can further include identifying asnon-confirming a tissue section if one or more mechanical damages arepresent in the tissue sample in the second imaging data but not in thefirst imaging data. In some embodiments, the method can further includeadjusting one or more operating parameters associated with removing ofthe tissue section to correct one or more mechanical damages. In someembodiments, the method can further include approving the tissue sectionif there are no mechanical damages are present in the tissue sample inthe first imaging data and the second imaging data. In some embodiments,the method can further include rejecting the tissue block if one or moremechanical damages are present in both the first imaging data and thesecond imaging data.

In some embodiments, one or both of the imaging steps compriseilluminating the tissue sample with UV light and imaging the tissuesample with a visible range camera to create the first imaging data orthe second imaging data. In some embodiments, the method can furtherinclude illuminating the tissue section to enhance a contrast betweenthe tissue sample and the embedding material in the tissue sample. Insome embodiments, one or both of the imaging steps can include imaging atissue section at one or more wavelength ranges, creating an imagingdata of the tissue section, segmenting the tissue sample from theembedding material based on a color and intensity information in thecolor imaging data, and identifying a size, a shape or edges of thetissue sample in the tissue section.

In some embodiments, the method can further include imaging the tissueblock, prior to removing the one or more sections, to generate abaseline imaging data of the tissue sample. In some embodiments, themethod can further include illuminating the tissue block with UV light.In some embodiments, the method can further include comparing the firstimaging data, the second imaging data or both to the baseline imagingdata. In some embodiments, the method can further include comparing anoutline, size, or shape of the tissue sample in the first imaging data,the second imaging data or both to an outline, size, or shape of thetissue sample expected from the baseline imaging data.

In some embodiments, a vision system is provided that include anillumination system configured to illuminate a tissue sample, an imagingsystem configured to create an imaging data of the tissue sectionilluminated by the illumination system, and a processor in communicationwith the imaging system to receive the imaging data and perform one ormore quality control analysis based on the imaging data. In someembodiments, the one or more quality control analyses are one or more ofa comparative analysis of the tissue section on a tissue block and aslide, an analysis of mechanical properties of the tissue section, ananalysis of sufficiency of the tissue sample, or an analysis of samplerepresentation on a slide.

In some embodiments, a histology system is provided that can include amicrotome configured to produce one or more tissue sections from atissue block, a transfer system configured to transfer the one or moretissue sections from the microtome to one or more slides, and a visionsystem. The vision system can include an illumination system configuredto illuminate a tissue sample, and an imaging system configured tocreate an imaging data of the tissue section illuminated by theillumination system. A processor is in communication with the imagingsystem to receive the imaging data and perform one or more qualitycontrol analysis based on the imaging data. In some embodiments, the oneor more quality control analysis are one or more of a comparativeanalysis of at least two of the tissue section on a tissue block, thetissue section on the transfer system, and the tissue section on aslide, an analysis of mechanical properties of the tissue section; ananalysis of sufficiency of the tissue sample; or an analysis of samplerepresentation on a slide.

The present disclosure also relates to system and methods for trackingand printing within a histology system. In some embodiments, a system isprovided that includes an information reader configured to readidentifying data associated with a tissue block, a microtome configuredto cut one or more tissue sections from the tissue block, one or moreslides for receiving the one or more tissue sections, and a printerconfigured to receive the identifying data and print, after the one ormore tissue sections are cut from the tissue block, one or more labelsfor the one or more slides, the one or more labels comprisinginformation associating the one more tissue sections on the one or moreslides with the tissue block.

In some embodiments, the system can further include a transfer mediumconfigured to transfer the one or more tissue sections from themicrotome to the one or more slides. In some embodiments, the transfermedium includes markings indicative of the identifying data for the oneor more tissue sections, the markings being configured to associate theone or more tissue sections with the tissue block. In some embodiments,the system can further include a transfer medium marking device to markthe transfer medium with markings indicative of the identifying data forthe one or more tissue sections, the markings are configured toassociate the one or more tissue sections with the tissue block.

In some embodiments, the system can further include a visualizationsystem configured to track the one or more tissue sections from themicrotome to the one or more slides. In some embodiments, thevisualization system is configured to make a comparison between the oneor more tissue sections on the one or more slides with the one or moreimages of the tissue block or the image of the section on the transfermedium. In some embodiments, the visualization system is configured tomake a comparison between the one or more tissue sections on the one ormore slides, on the tissue block or the transfer medium with a baselineimage of a tissue sample in the tissue block generated by imaging thetissue block with UV light prior to removing the one or more sectionsfrom the tissue block. For example, the comparison is based on a size,shape and outline of the tissue sample in the one or more tissuesections. In some embodiments, the visualization system is configured toread the one or more labels on the slide and confirm their associationwith the identifying data on the sample block. In some embodiments, theprinter prints the label individually for the one or more samples.

In some embodiments, a system can be provided that includes aninformation reader configured to read identifying data from a tissueblock, a microtome configured to cut one or more tissue sections fromthe tissue block, a transfer medium configured to transfer the one ormore tissue sections to one or more slides, and a printer. A processorcan be configured to receive the identifying data, cause the microtometo cut the one or more tissue sections, and subsequently cause theprinter to print one or more labels for the one or more slides, the oneor more labels comprising information associating the one more tissuesections on the one or more slides with the tissue block.

In some embodiments, the transfer medium includes markings indicative ofthe identifying data for the one or more tissue sections, the markingsbeing configured to associate the one or more tissue sections with thetissue block. In some embodiments, the system can further include atransfer medium marking device to mark the transfer medium with markingsindicative of the identifying data for the one or more tissue sections,the markings being configured to associate the one or more tissuesections with the tissue block. In some embodiments, the system canfurther include a visualization system configured to track the one ormore tissue sections from the microtome to the one or more slides. Insome embodiments, the visualization system is configured to make acomparison between the one or more tissue sections on the one or moreslides with the one or more sections on the tissue block. For example,the comparison is based on a size and edges of tissue in the one or moretissue sections.

In some embodiments, a method for tracking samples in microtomy isprovided that includes reading identifying data from a tissue block,cutting a first set of one or more tissue sections from the tissueblock, subsequently to cutting, printing one or more labels for the oneor more slides, the one or more labels comprising informationassociating the one more tissue sections on the one or more slides withthe tissue block, and transferring the one or more tissue sections toone or more slides and labeling the one or more slides with the one ormore labels.

In some embodiments, the method can further include comparing the one ormore tissue sections on the slides to the one or more tissue sections onthe block to confirm association of the one more tissue sections on theone or more slides with the tissue block. In some embodiments, themethod can further include cutting a second set of one or more tissuesections only after the first set of the one or more tissue sections isplaced on the one or more slides and labeled with the one or morelabels. In some embodiments, the method can further include comparingthe one or more tissue sections on the one or more slides with abaseline image of a tissue sample in the tissue block generated byimaging the tissue block with UV light prior to removing the one or moresections from the tissue block.

BRIEF DESCRIPTION OF THE DRAWINGS

So that those having ordinary skill in the art to which the subjectinvention appertains will more readily understand how to make and usethe surgical apparatus and systems disclosed herein, preferredembodiments thereof will be described in detail hereinbelow withreference to the drawings, wherein:

FIG. 1 illustrates an exemplary system for performing quality controlanalyses of a histology system;

FIG. 2 illustrates one embodiment of a lighting and imaging system toincrease the contrast of the tissue with respect to the paraffin;

FIG. 3 illustrates an exemplary tissue block showing a tissue inside anembedding material;

FIGS. 4A, 4B, 4C, 4D, 4E, and 4F illustrate a progression of images froma naive block to a fully faced block;

FIG. 5 is an exemplary flow chart illustrating a method for comparing animage of a tissue section with a baseline image of a tissue block;

FIG. 6 is a flow chart illustrating a method for making a facingdecision;

FIGS. 7 and 8 are images of a tissue ribbon under UV lighting;

FIG. 9 is a flow chart illustrating a method for making a facingdecision wherein the cutting ribbon and block face are imaged;

FIG. 10 is a flow chart illustrating various methods for block facing;

FIGS. 11A and 11B illustrate an exemplary image of a tissue embedded inan embedding material and a related exemplary graph showing therelationship between the tissue and embedding material;

FIGS. 12A and 12B illustrate an exemplary image of a tissue embedded inan embedding material and a related exemplary graph showing therelationship between the tissue and embedding material;

FIG. 13 illustrates an exemplary image related to laser dot diffusion;

FIG. 14 illustrates an exemplary graph related to column 2 dots sampledat 1 pixel across the block of laser dots on a tissue block;

FIG. 15 illustrates an exemplary image showing micro-serrations that ablade can leave on a tissue block;

FIG. 16 illustrates an exemplary system for performing tissue comparisonand mechanical quality control analyses of a histology system;

FIG. 17A is a section comparison of the sample block and the tissuesection on the glass slide;

FIG. 17B is a slide and paraffin block identification comparison;

FIGS. 18 and 19 are exemplary images of a tissue ribbon under UVlighting;

FIG. 20 is a flow chart illustrating a method wherein the cut sectionand block face are imaged;

FIG. 21A is a schematic view of one embodiment of the automated tapetransfer apparatus of the present disclosure illustrating the path ofthe tape, the apparatus having a barcode reader, in order to scan thelabel on the sample block, as well as the printed glass slide inaccordance with just-in-time printing, tracking, and identification ofcut tissue sections of the present disclosure; wherein a tape marking orprinting device laser etches or prints identifying marks onto the tapetransfer medium, and the glass slide is printed in real-time by aseparate slide printer.

FIG. 21B is a schematic view of one embodiment of the automated tapetransfer apparatus of the present disclosure illustrating the path ofthe optionally pre-printed tape, the apparatus having a barcode reader,in order to scan the label on the sample block, as well as the printedglass slide in accordance with tracking and identification of cut tissuesections of the present disclosure; wherein the glass slide is printedin real-time by a separate slide printer.

FIG. 22 is a flow chart illustrating one embodiment of the automatedsteps of the apparatus (system) having a barcode scanning, tape mediummarking, and slide printing system, to enable tracking of the tissuesection from block to tape to glass slide;

FIG. 23 is a workflow diagram of the section tracking system;

FIG. 24 illustrates an exemplary embodiment of an automated microtomydevice;

FIG. 25 is a schematic view of an exemplary embodiment of an automatedtape transfer apparatus illustrating the path of the tape, the apparatushaving imaging devices for taking images of the sample block and theslide;

FIG. 26 is a perspective view of a slide station of the automatedapparatus of FIG. 6;

;

FIG. 27 is a schematic view showing the tape prior to being applied tothe face of the sample block;

FIG. 28A is an elevated view illustration of a sample system layout inaccordance with some embodiments of the present disclosure;

FIGS. 28B and 28C are isometric view illustrations of a sample systemlayout in accordance with some embodiments of the present disclosure;

FIG. 29 is a flow chart illustrating the processing of tissue blocks inthe automated tissue sectioning system in accordance with someembodiments of the present disclosure

FIG. 30 is a flow chart showing illustrating the automated steps of thesystem of FIG. 8

FIG. 31 is a flow chart illustrating one embodiment of the automatedsteps of the apparatus (system) having an imaging device to determinewhether tissue sections are sufficient for transfer to slides;

FIG. 32 is a flow chart illustrating the automated steps of an alternateembodiment of the apparatus;

FIG. 33 is a flow chart illustrating the automated steps of an alternateembodiment of the apparatus; and

FIG. 34 is an exemplary embodiment of a computing device.

DETAILED DESCRIPTION

The present disclosure provides systems and methods for various qualitycontrol analyses on the tissue samples during microtomy. An exemplaryembodiment of systems and methods for quality control of a tissue sampleare shown in FIG. 1. Such systems can be configured to provide varioustypes/aspects of quality control 100 including printing and tracking102, tissue facing analysis 104, and mechanical tissue integrityanalysis 106.

As shown, a tissue section can be cut from a tissue sample, such as asample block. Various quality control analyses can be performed relatedto the tissue section to determine the integrity of the tissue section.As will be explained in more detail below, one or more analyses can beperformed as the tissue section is being transported from the sampleblock to a slide or other medium or can be performed once the tissuesection is located on the slide or other medium. The process can beperformed manually or with an automated system.

Various images can be taken of the tissue block and tissue sections forcomparison to perform the various quality control analyses. For example,images can be taken, including but not limited to, an image beforetissue sectioning to create a baseline image of the block, a face of thetissue block, a tissue section after being cut from the tissue block, atissue section as it is being transported via a transport system ortape, and a tissue section positioned on a slide. Comparing any of theseimages can be used to determine if the tissue section is sufficient andbe used once positioned on the slide. If a comparison reveals any issuesor errors, the tissue slice can be discarded, the tissue block can bediscarded, and/or an adjustment can be made to any of the physicalcomponents of the system, depending on the types of issue or error thathas been identified. Various operating parameters can be adjusted basedon the type of the mechanical damages or defects detected using thevision system of the present disclosure. Such operating parametersinclude, but are not limited to, sharpening or replacing the microtomeblade, adjusting hydration time or temperature, replacing the transfermedium, or adjusting the operating parameters of the transfer medium(for example, speed) or pressure applied to the transfer medium againstthe tissue sections.

In some embodiments, analysis is performed that compares the tissuesection to the sample block to match the tissue section to the sampleblock. In some embodiments, analysis is performed is determine acondition of the tissue section, either during transport or after slideplacement. Additional analysis of the tissue section can be performed,as will be discussed in more detail below. Further, various methods canbe used to perform quality control, including but not limited to the useof a visualization/imaging system that can be configured to image one ormore of the tissue sample block, the tissue section during transport,and the tissue section on a slide such that the images can be used toperform one or more of the quality control analyses.

In some embodiments, histology systems can provide tracking andcomparative analysis of cut tissue sections in a manual or an automatedtissue transfer apparatus/system. The decision system analyzes/comparesone or more parameters or characteristics/features of the cut tissuesection on the microscope slide and the sample block. Alternatively, orin addition, the system analyzes/compares one or more parameters orcharacteristics/features of the cut tissue section on the microscopeslide and the cut tissue section (slice) right after it is cut from thesample block. These systems ensure that the sections on the glass slideare properly matched to the tissue sample block. This is the comparativeaspect of the quality control system that improves sample tracking inthe laboratory.

In some embodiments, a system is provided that can provide feedback toenable self-correction or adaptation. This feedback system providesanother aspect of quality control.

In some embodiments, the systems also provide feedback systems ofquality control. In some embodiments, for example, once the qualitycontrol system sends a flag, a feedback process could be triggered, andas a result various actions can automatically be taken without humanintervention. For example, if there is a flag, e.g. a mismatch, examplesof downstream actions that can occur can include recalibration ofinstruments or of algorithms, thus permitting the system/process toself-correct or adapt. In some embodiments, the feedback process caninteract with the block facing decision. In a quality control processwhen a defect is detected, a root cause is searched to fix the issue. Assuch, when a defect is detected by the quality control process, apredetermined set of possible root causes will be checked. For example,if there are bubbles under the tissue section on the glass this couldmean the transfer medium (tape) to glass applicator roller has amalfunction and the user can be warned about this. Or if the tissue isshredded in the auto QC images, the system would force a new sectioningblade exchange at the microtome. Once the root cause is identified oneor more operating parameters can be changed as explained above.

For example, in an exemplary embodiment of a feedback system, a QCsystem suddenly starts generating an increased number of flags forsection quality one day. This can indicate that a condition in thesystem has changed and that corrective action is needed. An increasedmismatch rate can trigger a suite of self-tests, and one of thoseself-tests could then trigger a correction mechanism (e.g., replace themicrotome blade). By way of another example, supposing it is noted thatthe tissue starts being absent on slide at a high rate. This could meanthat the facing algorithm is failing or that tissue is falling off ofthe tape, and again self-tests could trigger corrective actions. It willbe understood that these are just provided by way of example as otheractions in response to various events are also contemplated in thequality control feedback systems.

It should be appreciated that the feedback loops can be used with theother quality systems disclosed herein.

It will be understood that any transfer medium (also referred to astransport medium) other than tape can be utilized. Therefore, referencesto tape herein are used for convenience as the systems and methodsdisclosed herein are fully applicable to other transfer medium not justtape.

Sample Preparation

The sample can be a tissue, organ, organism, frozen liquid, or otherbiological sample. In some embodiments, the sample can be pre-stained orpre-treated in some other manner to facilitate facing, as will bediscussed in more detail below. The process of initial preparation ofthe sample can include, in some embodiments: a) transfer of thebiological tissue when removed from an organism into a fixative, e.g.,formalin container; b) after fixation, the tissue is transferred into alabelled tissue cassette; c) after transfer to the cassette, the tissueis processed by i) dehydration via immersion in alcohol to remove waterand formalin, ii) clearing via a solvent to remove the alcohol, and iii)applying an embedding material, such as paraffin wax, to surround thetissue in a large block of molten material to create the “sample block.”The paraffin is poured over the dried and chemically treated tissue in amold. In some cases, the histotechnologist presses on the tissue duringthe molding process but there is always paraffin between the bottom ofthe mold and the tissue sample. When the block solidifies it provides asupport matrix during the tissue sectioning process. That is, when theparaffin is cooled down, it gets removed from the mold so it is in theform of a paraffin block with tissue embedded in it. The molded tissueparaffin combination is supported on a plastic cassette. The plasticcassette provides the features for the tissue to be held in a microtomeclamp. On the opposite side of the cassette where it is clamped theparaffin/tissue combination is cantilevered. This side corresponds tothe bottom of the mold in the previous step. The excess paraffin due tomolding needs to be removed to expose the tissue so tissue sections canbe identified and analyzed/evaluated. The thickness of the excessparaffin varies greatly from tens of microns to a few hundredmicron-meters.

The plastic cassette holding the tissue in the paraffin mold could havedifferent colors. In some embodiments, the quality control systemdetects a color of a cassette holding the sample block to cross-check asample type. In some laboratories they assign these colors to certaintissue types. For example, the lab may choose to use pink cassettes forbreast tissue. When the system detects a certain color that issignificant to the lab it could cross check the sample type with the labdata to ensure the colors match. This provides an additional backup ofthe quality control system. Also, this data can be used during imageprocessing so that variations in image background is effectivelyfiltered out.

When the tissue block is placed in the chuck for cutting (sectioning),the paraffin side faces the cutting blade of the microtome, but themolding process is not perfect, and the tissue is not on the surface ofthe paraffin but under a paraffin layer. The new block is firstsubjected to sectioning with relatively thick sections to remove the 0.1mm-1 mm layer of paraffin wax on top of the tissue sample. After removalof this superficial paraffin layer and when the complete outline of thetissue sample is exposed, then the block is ready to be sectioned. Oncethis paraffin layer is removed, in clinical and research settings, thetissue is typically sectioned to 3 μm to 5 μm thickness. This processfor removing this paraffin layer and exposing the large cross section ofthe tissue is referred to as block facing. After removal of thissuperficial paraffin layer, the tissue sample is exposed and ready to besectioned and put on the tape for transfer to a glass slide foranalysis, e.g., pathology or histology. That is, when enough paraffinhas been removed (the block is referred to as “faced”), subsequent blocksectioning provides tissue sections for placement on glass slides foranalysis (processed further for evaluation).

Note that although paraffin is described herein as the embeddingmaterial, it should be appreciated that other embedding material couldbe utilized, including frozen sections.

Visualization System

The histology system includes, in some embodiments, a visualizationsystem that can include an illumination system and an imaging system.The illumination system aids in imaging/discernment/differentiation ofthe tissue and paraffin which can then be imaged by the imaging systemfor evaluation. Thus, the differentiation of the tissue from theparaffin is enhanced for the images taken on the sample block, tape,and/or slides. Various illumination systems and imaging systems arediscussed below. In some embodiments, the visualization system utilizesappropriate optics (illumination system/method, imaging system/method,detection system/method) to be followed subsequently by computationalprocessing. This computational processing provides comparativeassessment of images for quality control, as will be described in moredetail below.

It should be understood that the term “image” includes data in any formor format generated by the imaging system that is representative of animage or can otherwise be analyzed to determine information about thesubject being imaged. The images taken by the visualization system canbe used such that the images themselves can be processed, analyzed,and/or compared for quality control analyses, or any data representativeof the image or the subject being imaged, including data for creatingthe image or data representative of the image or the subject beingimaged, can be used.

In some embodiments, the visualization system can be configured to a)take an image of i) the tissue sample block containing tissue embeddedin an embedding material, ii) a block face of the tissue sample block,iii) a tissue section after cut from the sample block, iv) a transfermedium carrying the cut tissue section or v) a slide containing the cuttissue section; and b) take an image of the cut section from the sampleblock either on a slide or a transfer medium (e.g., tape). In someembodiments, this can be done to enhance differentiation of tissue fromthe embedding material in which the tissue is embedded for enhancingimages taken by the imaging device.

FIG. 2 illustrates an embodiment of a visualization system 110 thatincludes an illumination system 112 and an imaging system 114 that canbe used to image one or more of the tissue block, a tissue sectionassociated with a transport system, and a tissue section associated witha slide. As shown, the illumination system 112 illuminates the sampleblock 116 supported on the sample supporting chuck 118. Note referencenumeral 122 illustrates the sample block under high contrast lightingwhere the contrast between the paraffin and tissue is pronounced;reference numeral 120 on the other hand illustrates the sample blockunder low contrast lighting where it is more difficult to differentiatethe tissue and paraffin. The illumination can alternatively or inaddition illuminate the tissue section after cut from the block. Varioustypes of illumination systems are described below. An imaging device,e.g., a camera, takes images of the sample block 116 for evaluation.Classical image processing techniques, as described below, can beutilized for evaluation. Recent Artificial Intelligence based imageprocessing techniques can also be utilized where the teaching andtesting phases are all the done at the development site.

As shown, the imaging system 114, e.g., a camera, images the block faceof the sample block 116 as the illumination system 112 illuminates theblock face such that the tissue to paraffin contrast is increased. Thisimaging system in some embodiments can be triggered by the software orhardware to take consecutive images of the block 116 as it is sectioned,done by non-human intervention. Classical image processing techniquesbased on edge detection (finding the boundaries of the object within theimages by detecting discontinuities in brightness), color, hue, orintensity tracking in consecutive images, or similar attributes of eachpixel and their variation in the context of the image can be utilized.The imaging system can be part of the automated sectioning system, andthe automation system has the capability to synchronize imaging with theblock sectioning whether through software operations or through hardwaretriggers such as position sensing switches. As noted above, images canalternatively or in addition be taken of the cut tissue section aftercut by the microtome.

In some embodiments, line scanning is utilized. Line scanning involvesbuilding the image one line at a time using a line sensor passing inlinear motion over an object. Thus, in this method, the area of theblock-tissue ribbon interface at the blade during cutting is scannedafter the tissue block is cut by the blade. The software then stitchesthe lines and creates an image of the section cut. The line of sectionright over the blade can be imaged and a 2D image can be constructedfrom a series of line images. The line imaging device in someembodiments can be placed inside the roller for line scanning as thetape moves past the roller or as the roller moves along the tape. Insome embodiments, the imaging device can be placed outside the roller.In either case, the imaging can be tied to tape transport with thetransmitted light on one side of the tape. The illumination device couldbe inside or outside the roller. Several advantages of line scanningcompared to taking a 2D image of a tissue section already cut include i)the reconstructed line scanned image will be a “flat” representation ofthe tissue face and will not have a risk of being wrinkled or rolled;ii) it is easy to determine the boundaries of the section beginning andend; and iii) all parts of the image will be in focus because the bladeand the cutting forces provide a consistent location for the tissuesection.

The system can utilize a one-dimensional scan across the sample, aprofile or an image all at varying degree of sampling resolution.

Imaging the tissue section on the slide can also provide downstream usefor digital pathology. In scanning the tissue in a slide scanner andprocessing it computationally, it is beneficial to first determine wherein the slide the tissue is sitting (geometrically). This is beneficialfor the auto-focus algorithms in the scanner, or to reduce computationalsteps in the computer-aided diagnostics where tissue is segmented. Thesystems described herein, e.g., UV illuminated fluorescent mode, provideclear visibility to permit easy segmentation. Such segmentation/locationinformation can be passed back to the laboratory information system tobe integrated downstream with a scanner or a computer aided diagnosticplatform, when the slide with that tissue section is being used.

In some embodiments, illumination and signal capturing using RamanSpectroscopy can be used to identify the paraffin matrix over thetissue. Raman spectroscopy can be used to determine vibrational modes,rotational modes or other low frequency modes of molecules to provide astructural guide to identify molecules. As the light excites themolecules, the molecules will reflect light in a different wavelength,and the wavelength thereby allows detection of the composition. That is,Raman spectroscopy detects certain materials based on the scatteredlight quantitatively. The tissue itself is impregnated with paraffin butthe density is less than the paraffin in other areas of the block.Progressive image captures with Raman spectroscopy of the block aftereach section gives a quantified metric utilized for the comparison. Thismethod could be used in parallel with visible light imaging to determinethe tissue area a priori which would increase the efficiency of theimplemented method.

In some embodiments, the imaging system comprises a sensor and aradiation system. The method/system captures signals from the paraffinportion of the tissue block or cut section such that the level of thesignal decreases when the paraffin over the tissue area gets thinner.This is due to the fact that different materials absorb differentradiation wavelengths, and tissue and paraffin radiation absorptionwavelengths are different. As the paraffin layers over the embeddedtissue are removed, the absorption spectra (e.g., IR absorption spectra)will change. By tracking this change over consecutive images, theabsorption levels correlate with the amount of paraffin over theembedded tissue. In this manner, the amount (or removal) of paraffin canbe determined by comparative or computational processes.

For the tissue on the tape or on the slide, either reflection-mode ortransmission-mode may be used. It may be beneficial to utilize thetransmission mode in terms of location of the light sources and cameras.In particular, the imaging of the section on tape can be done byembedding the light source or the camera inside a transparent roller (ora roller with a transparent window), using either a line source or aline camera.

The illumination system can emit light in UV, near IR, IR, orvisible/broadband ranges, for example. The emitted light may havedifferent colors. In some embodiments, the illumination system caninclude LEDs, OLEDs, lasers, light bulbs or similar light emittingdevices or materials. As also shown, the imaging systems can includevarious areas to take images including the tissue block or the tape oron the glass slide. A computation system processes the images, forexample quantitatively, for identity tracking.

It should be understood that these various illumination and imagingsystems described herein, and various associated methodologies, areprovided by way of example as other illuminations systems can beutilized to enhance differentiating the tissue and the paraffin (orother embedding material), and other imaging systems can be utilized andother computational systems or comparative processing systems can beutilized for identity tracking and sample integrity checking. Also, anycombination of the illumination systems and/or any combination of theimaging systems can be utilized.

Light (coherent or non-coherent) can be used via absorption, refraction,scattering, raman scattering, fluorescence, phosphorescence,interference and wavelengths can be continuous or discontinuousdistributions anywhere in the spectrum from x-ray to radio waves or anycombination of these modalities. Transmission of reflectance mode can beutilized.

In some embodiments, the imaging system can include a plurality ofimaging devices for imaging the sample block for quality control asdescribed herein. The imaging devices can work in unison to create asingle 3D image of the sample block. Various types of imaging devicescan be used, including but not limited to a visible light camera,spectrometer, a multispectral camera, a hyperspectral camera, a mid-waveinfrared (MWIR) camera, and a Raman spectroscopy camera.

Hyper-spectral imaging of tissue in paraffin blocks allows discernmentof the unique absorption of paraffin to be identified separately fromthe various tissue reflection peaks in a single image. With ahyperspectral cube based on 3-5 nm steps in frequency the specificreflectance peaks of paraffin and tissue can be isolated and the depthof the tissue can be identified.

Multi-spectral imaging utilizing an array or series of illuminationsources from the UV through to the infrared can allow for theinterrogation of tissue and paraffin at unique spectral lines withmultiple exposures at different color frequencies that elicited specificabsorption or reflection characteristics of the tissue in question.

In some embodiments, the tissue block may be illuminated with astructured light and the returned light can be used to determine variouscharacteristics of the tissue block, the tissue sample of both. Forexample, the outline or cross-sectional area of the tissue sample or thedepth profile of the tissue block can be determined using the structuredlight. In some embodiments, the depth profile is the thickness of theembedding material to be removed. In some embodiments, the removal ofsuch thickness can expose the tissue sample to a pre-determinedcriteria. In some embodiments, the structured light refers to anillumination of the tissue block in a specific pattern. In someembodiments, the structured light may be spatially structured, that is,the tissue is illuminated in a geometrically structured pattern, such asa grid, stripes, concentric circles, etc. In some embodiments, thestructured light may be spectrally structured, that is, the tissue issimultaneously illuminated by light having different wavelengths. Insome embodiments, the wavelength may be selected from differentintensities, bands or colors. In some embodiments, the spectrallystructured light can be in the same or predominantly the same intensityrange (for example, UV), but have different specific wavelengths withinthat intensity range. In some embodiments, the spectrally structuredlight could constitute light predominantly from one or more frequencybands, such bands tailored to the optical properties of tissuemolecules, such optical properties including, for example, fluorescenceabsorption and emission spectra. As an example, a wavelength range withpredominantly UV radiation could give rise to strong autofluorescencefrom certain tissue compartments, facilitating the subsequent processingsteps in the invention.

Laser dot diffusion can also be used. A green laser source at 515 nm anda Diffractive Optical Element (DOE) can be used to create an array oflaser dots on the paraffin tissue block thereby sampling the dispersionacross the whole of the block in a single image. Using different DOEsdifferent size laser arrays can be projected on the surface of theblock. Alternatively, one can move the block and take one or more imagesto increase the laser dot density on the surface of the block. The imageof the tissue block is taken while it is illuminated with the laser DOE.One or more channels of this image can be analyzed to determine thelight scatter. For example, when the green laser source is used toilluminate the block, the green channel would have the highest response.One can also use the gray value of the image for the analysis.

In some embodiments, multiple cameras are provided adjacent the blockface for capturing the images of the block face simultaneously. Thecameras and their software can use inherent geometric features of theblock or the mechanisms around the block to orient each image to thesame reference geometry. The images from multiple cameras can be used toconstruct a 3D image of the tissue in the block. Such a 3D image wouldincrease the speed of facing the block because the microtome canpredetermine how much to face the block.

The images from multiple cameras can also be used for enhancing the 2Dimage quality by merging multiple images taken from different angles.The clarity of images would increase when more images are processed tocreate a single image. This can be used for image enhancement of thesample block face, cut tissue section and/or the slides having the cuttissue sections deposited thereon.

Another way to enhance imaging is to mount the imaging device, e.g.,camera, on a computer-controlled motion stage, the stage movable to avariety of positions. In this manner, a plurality of images of thesample block can be taken from different positions along the motion pathof the stage. Alternatively, the block or the tissue on the glass slidemay be moved with respect to the camera to have a similar effect. Thus,the relative motion of the imaging device with respect to the block orwith respect to the sample can be utilized, with relative movementdenoting movement of the imaging device, movement of the block or glassslide, or movement of both the imaging device and the block or glassslide. The images could be processed to create a single image or selectimages utilized for comparative analysis.

In some embodiments, the imaging equipment, e.g., the digital cameras,are housed within a closed chamber. Within the closed chamber, radiationsources with controlled polarity and wavelength can be provided toilluminate the biological tissue sample inside the enclosed chamber toenhance imaging. The radiation sources can have dynamically adjustablepolarity and wavelength while the biological sample is being imaged.Such illumination can be provided for the tissue on the sample blockand/or the cut tissue section on the slide. The system can includevarious software algorithms for performing different analyses. Forexample, an algorithm can be utilized to determine the depth of thebiological tissue sample under the paraffin, an algorithm can determinethe whole or partial 3D shape of the biological tissue embedded in theparaffin and/or an algorithm can determine the largest surface areacross section of the biological tissue and the depth of said crosssection. Another algorithm which can be provided is to extractbiological tissue physical properties from the images.

For example, the algorithm could determine physical properties includingbut not limited to largest tissue outline, number of pieces of tissuesin the sample (paraffin) block, and depth of each piece in the block. Insome uses, the paraffin block may have multiple pieces of tissue in it.These may be the same tissue cut into multiple pieces or multiple tissuesamples collected at multiple points from the patient. Placing multipletissue pieces in the same block helps laboratories decrease the tissuestaining cost. The feature (algorithm) makes sure all the tissue piecesare cut in the same section, that is, they are in the same plane.

In some embodiments, for comparative analysis, a projection systemprojects an orientation pattern on the block face and the same patternis projected on the slide. A software algorithm can be used to determinethe relative orientation of the biological tissue sample in the paraffinblock and the tissue on the slide.

The quality control system of identification matching can be used withany of the apparatus/systems and methods described herein.

Tissue Block Analysis

In some embodiments, one or more images can be taken before tissuesectioning of a complete tissue block. In some embodiments, across-sectional image can be taken at a known depth into the tissueblock to estimate the location, size, and shape of the tissue within theblock. One or more of these images can be used to create a depth profileof the tissue embedded in the tissue block. In some embodiments, animage of an estimate of the composition of the entire tissue block canbe created. The tissue block can be illuminated, using the visualizationsystem as described above, such that the illumination light causes thetissue inside the tissue block to fluoresce. While various illuminationtypes can be used, in some embodiments, the tissue block can beilluminated with UV light such that the UV penetrates through theembedding material around the tissue and causes the tissue sample buriedin the embedding material to fluoresce. Thus, the tissue embedded in theembedding material can be fully outlined within the block. This canprovide as baseline image that can be used for various comparisons toexposed tissue on the face of the tissue block, on the transport system,and on the slides after transport. An exemplary baseline image of anentire tissue block 132 is shown in FIG. 3. As shown, the baseline image130 includes a tissue sample 134 embedded in an embedding material 136to form the tissue block 132.

This baseline image of the total composition of the tissue block can beused in a variety of the methods disclosed below. For example, whendetermining whether the tissue block is faced, an image taken of theblock face can be compared to the baseline image. Thus, a comparison canbe made between the “faced” area of tissue and the “buried” area oftissue from the baseline image as it related to block facing.

The baseline image can also be used for other quality control analyses.For example, images of tissue sections cut from the tissue block, tissuesection on a tape, or tissue sections on a slide can be compared to thebaseline image. The tissue sections can be compared to the “buried” areain the baseline image to determine integrity of the tissue section,completeness of the tissue section, and/or physical properties of thetissue section or confirm that the tissue section originates from aspecific block.

For example, as is described in more detail below in connection withFIGS. 4A-4F, the initial sections will only have the embedding material(FIG. 4A), but subsequent sections (FIGS. 4B-4F) will include a tissuesample and the outline of the tissue samples in the sections can becompared to the expected outline from the baseline image for facing,tracking and mechanical integrity decisions. In some embodiments, thesystem can determine an outline of the tissue on the sample block and anoutline of the cut tissue section on the transfer medium or on the slideto determine if a match is present.

Block Facing Methodologies

In some embodiments, systems and methods are provided for facing ofbiological sample tissue, including facilitating and improving the blockfacing decision to determine when the tissue layer, embedded in paraffinor other embedding material, has been reached, i.e., detection of fullfacing of the tissue block. In some embodiments, this can includedecision systems and methods for tissue block facing assessment anddetection in an automated apparatus. The systems/methods increase thespeed of sectioning and the quality of the eventual sections. In someembodiment, facing the tissue block relates to the amount of embeddingmaterial that needs to be removed. For example, a depth profile of thetissue block can be used, such that the depth profile relates to athickness of embedding material that can be removed to exposed thetissue sample to a pre-determined criteria that relates to a distancebetween the surface of the embedding material and the tissue sample. Forexample, the pre-determined criteria can be an amount, or depth, ofmaterial such that when removed from the face of the block can reach asurface of the tissue sample, or the pre-determined criteria can beamount of material such that when removed a sufficient cross-section ofthe tissue sample on the block face is revealed. The cross-sectionalarea of exposed tissue can vary. In some embodiments, the criteria canbe between 20% and 60% of the cross-sectional area of the tissue sample.In some embodiments, the criteria can be between 20% and 80% of thecross-sectional area of the tissue sample. In some embodiments, thecriteria can be between 40% and 60% of the cross-sectional area of thetissue sample. It will be understood that the amount of cross-sectionalarea of revealed tissue sample can vary and be any amount.

In some embodiments, image-based decision systems and methods for tissueblock facing assessment and detection can be used. A machine visionsystem can be used for facilitating and improving the block facingdecision to determine when the tissue layer, embedded in paraffin, hasbeen reached, i.e., detection of full facing of the tissue block. Thismachine vision system for automating the block facing decision hasapplication in automated transfer of cut tissue sections to supporting(carrying) media, e.g., tape, for subsequent transfer to microscopeslides. In some embodiments, a system and method of computationalprocessing is provided after the visualization system utilizing opticsas disclosed herein is operated. Thus, the visualization system utilizesappropriate optics (illumination system/method, imaging system/method,detection system/method) to be followed subsequently by computationalprocessing. This computational processing provides comparativeassessment of images to determine the status of the block facing.

In some embodiments, the illumination and imaging systems can be used toincreased contrast between the tissue and paraffin sections of thetissue block, as shown in the progression of images of FIGS. 4A-4F. Forexample, the illumination system can emit illuminating wavelengths toenhance the contrast between the tissue and paraffin. In someembodiments, this allows the system to determine a status of facing of ablock containing a sample of tissue embedded in an embedding material.The method comprises the steps of a) increasing a contrast of the tissueand the embedding material to enhance differentiation of the tissue andembedding material, which can include the step of illuminating one ormore of i) a block face of the tissue sample block or ii) the sectiontransport medium or iii) the slide, (for example, utilizing UVradiation); b) imaging the tissue and embedding material; c) processingthe images to assess when the block is faced; and d) after the block isfaced transferring the tissue to a tissue carrying medium. Theprogression shows five images by way of example to illustrate theprocess from the initial naive block at image 1 to the fully faced blockat image 5. While FIGS. 4A-4F illustrate a series of 6 images, it willbe understood, however, that any number of images can be used to achievethe objectives. Six images are shown for ease of description. Morespecifically, in the images shown in FIGS. 4A and 4B, the target tissuesection is fully covered by the paraffin, so the first n-sections willbe have to tissue sample, followed by section with a faint outline oftissue discernable. In the image shown in FIG. 4C a section of theparaffin has been removed but the tissue is still covered by theparaffin, although a larger tissue outline is shown, and in the imageshown in FIG. 4D, more of the paraffin has been removed but there is astill a thin layer of paraffin over the tissue In the image shown inFIG. 4E, the tissue section is shown within the white outline, with theparaffin still around the edges (outside the boundary/periphery definedby the outline) and in the image shown in FIG. 4F the fully faced blockis illustrated with the entire tissue section exposed. Note the paraffinblock is shown schematically as having a planar top (exposed) surface,however, it should be understood, that the block does not necessarilyhave a flat surface as shown.

The facing decisions can be achieved using a manual or automated system.In some embodiments, an automated system for determining a status offacing of a block containing a sample of tissue embedded in an embeddingmaterial is provided in an automated apparatus and includes an imagingdevice for differentiating the tissue from the embedding material suchas paraffin, and in response to the differentiating of the tissue andembedding material, the apparatus determines when the block is fullyfaced and in response to determining when the block is fully faced theapparatus automatically cuts and transfers sections of tissue from theblock for subsequent analysis. In some embodiments, after determiningfacing of the block is complete, the system automatically stops facing.In some embodiments, after determining facing of the block is completeand the system automatically stops facing, the cutting and transfer oftissue sections to tape automatically starts, and the tissue sectionscan be mounted on slides.

Various parameters/characteristics can be used to assess the status offacing of the block and make a determination about whether or not theblock is fully faced. In some embodiments, a characteristic of thesample block or the tissue section can be determined after being cutfrom the block. In some embodiments, an evaluation, e.g., measurement,can be made of the sample block itself or the cut tissue section itself.In some embodiments, a comparative analysis of the sample block or cuttissue section can be made to a prior evaluation, e.g., measurement. Insome embodiments, the sample block can be cut to a predetermined depth.It will be understood that any combination of these variousparameters/characteristics can be used to make the facing determination.

In some embodiments, an automated system is provided toenhance/facilitate the block facing decision in an automated tissuetransfer apparatus where the sample block is sectioned, the tissue istransferred to tape or other media and transferred to a glass substratewhich is suitable for analysis under a microscope after furtherprocessing, as will be discussed in more detail below.

The sample block can be examined from various angles such asperpendicular to the front face, at a glancing angle, perpendicular tothe side face or at a glancing angle or any combination of these.

Block measurements can be used or just cut sections can be used, or bothcan be combined.

In some embodiments, automated determination/decision making for blockfacing is achieved by enhancing differentiation between the paraffin andtissue and determining via the imaging system when block facing iscomplete. In some embodiments, this can be achieved using theillumination system and imaging system described herein. Theillumination system can aid in discernment/differentiation of the tissueand paraffin which can then be imaged by the imaging system forevaluation. Thus, the differentiation of the tissue from the paraffin isenhanced, and the images taken, on the block, holder, tape, and/or otherlocations. For example, the machine vision system can enhance theassessment of when the tissue layer, embedded in paraffin, has beenreached, i.e., when thick sections of paraffin wax on top of the tissuesample on the sample block have been removed to expose the tissue sampleso the tissue sample can be sectioned, transferred to a glass slide andprocessed further for evaluation.

As shown in FIG. 5, in some embodiments, the baseline image of thetissue sample can be used to assist in the facing decisions. Inparticular, in step 140, a cross-sectional area of buried tissue can bedetermined using UV fluorescence to create the baseline image. In step142, as tissue sections are being removed from the tissue block, a facedarea of the tissue can be determined using one of many alternativemethods. In step 144, the baseline image and the image of the block facecan be compared to make a judgment as to facing. For example, as shownin FIGS. 4A-4F, as progressive sections are removed from the tissueblock, the outline of the tissue sample in these sections willprogressively change. In some embodiments, the system can decide whenthe facing is complete based on the expected outline of the tissuesample based on the knowledge of the baseline image. For example, thefacing can be completed when only the top portion of the tissue isreached. Alternatively, the facing may be completed when the mid-sectionof the tissue sample is reached. Such decisions can be made by comparingthe outline of the tissue sample in the sections to the expected outlineof the tissue sample from the baseline image. In step 146, confirmationof the buried-tissue area to the section-on-tape area (for example,using UV illuminated image on tape) to confirm the facing judgment madein step 144.

In some embodiments, as shown in the flow chart of FIG. 6, a UVmethod/system can be utilized for the facing decision. The steps are asfollows: i) illumination by a pre-selected range of wavelengths (e.g.,the UV range) (step 150); ii) using appropriate optics in step 152 tocreate images on a color camera, i.e., a camera that simultaneouslytakes images in multiple wavelength ranges such as an RGB camera forexample; iii) using color and intensity information from the resultingimage to segment out the portions of the block where tissue resides instep 154; iv) monitoring the size of the tissue region, and the edges ofthe tissue region in step 156, as the facing cuts occur; and v)detecting an appropriate change in the quantities monitored in step (iv)leading to the “facing decision” in step 158. As discussed above, thesesteps are automated and do not require user input during the process.Note that intensity in a gray scale can be used as alternative to colorimages. FIGS. 7 and 8 are exemplary images of a tissue ribbon under UVlighting.

In some embodiments, as shown in the flow chart of FIG. 9, UV radiationor other wavelengths can be used to enhance the tissue/paraffincontrast, but observations are made of both the block face and the cutribbon. After applying illuminating wavelengths of light in step 160,the imaging system creates a color image of the block face (step 162)and of the cut ribbon (step 164). In this method, first appearance oftissue-fluorescence in a cut section signals that “facing” has occurred.This can be done either based on the total fluorescence from the ribbon,in step 166, (so that there is no need to fully image the ribbonface-on) or alternatively, the ribbon could be imaged face on, in step168. Such imaging could be facilitated in some embodiments by having theribbon on the tape and comparing the image of the ribbon with the imageof the block face (each with UV illumination). The images are processedand evaluated for tissue segmentation (or othercharacteristics/parameters described herein), in step 170, as the sizeand edges of the tissue regions can be assessed and quantified (step172) to detect the changes to determine the status and completing offacing (step 174). In another alternate method, the imaging is done onlyon the cut ribbon e.g., when on and/or off the tape as an alternative toblock imaging instead of as a supplement.

The flow chart of FIG. 10 illustrates various systems that can beutilized by way of example for determining block facing of a naive orpartially faced block (step 180) or a fully faced block (step 182).

There are a variety of ways to cut into the tissue block to expose thetissue embedded in the paraffin or other embedding material. Evaluationof the tissue can be performed after cutting from the block, of thesample block itself, or both. In some embodiments, a predeterminedamount of paraffin can be removed from the tissue block to exposetissue. For example, with a naive or partially faced block (the lattercan be the result for example from a failure of the facing procedure ina previous run), determination of a faced block can be made by taking Nsections at Mum thickness for removal of a fixed/pre-programmed depth ofparaffin (step 184). After such cutting, the block facing is consideredto be completed (step 186).

In some embodiments, an evaluation of the tissue block is made after aseries or one or more cuts are made into the tissue block to determineif the block is faced after each cut. The sections are cut from theblock and through imaging of the block, the cut tissue section and/orthe cut tissue section on the tape (or other transfer media), or othertechniques, the sections are evaluated until a determination that theblock is fully faced is made For example, X sections at Y um thicknessare taken (step 190), then a section is cut from the block (step 192)and an evaluation of the cut section is made (step 194) to confirm theblock is faced (step 196). This check could be by taking images of thecut tissue section or the cut tissue section on the tape (or othertransfer media).

Evaluation, e.g., measurement, of the sample block and/or cut tissue (onor off the tape) (step 202) can be made with each cut section (step 200)so it leads to a decision to continue sectioning (not yet faced) or stopsectioning (fully faced as in step 204). In some embodiments, thedecision to continue sectioning can lead to a decision to cut severalsections before evaluation, e.g., measurement. In some embodiments,several consecutive sections can be cut before leading to an evaluationor decision.

In some embodiments, the decision can be made based on evaluation of thesample block and/or specific cut section (on or off the tape). In someembodiments, the decision can be made by comparing a second measurement(or other criteria or parameter) to a first measurement (or othercriteria or parameter) in accordance with an algorithm. The decision canalso be made based on a fixed depth rather than an evaluation of thespecific block face or cut section

In some embodiments, depth measurement can be used to determine tissuedepth under the embedding material, such as paraffin, (a priori)(step210). In some embodiments, the tissue depth can be determined from thebaseline image, as for example shown in FIGS. 4A-4F. A 3D imagingtechnique can be used for example to determine tissue depth without anyfurther measurement. The sample block is cut to the measured depth toexpose the full face of the tissue in step 212. At this time, the blockcan be considered as fully faced (step 214). In some embodiments, afterthe sample block is cut to the pre-determined depth, additional image(s)can be taken, e.g., images of the cut tissue section on the tape (orother transfer media) to confirm the full face is reached, in steps 216and 218. In some embodiments, depth measurement can be used to determinetissue depth under the embedding material in step 220. After the sampleblock is cut to the pre-determined depth in step 222, a section is cutfrom the block (step 224) and evaluated to confirm the block is faced instep 226, e.g., by images of the cut tissue section or the of the cuttissue section on the tape (or other transfer media). If not fullyfaced, another section is cut from the block and checked. This continuesuntil a determination that the block is faced, in step 228. It will beunderstood that other methods can be used to determine depth, includingbut not limited to ultrasound, X-ray imaging, and other non-light baseddetection methods, and comparison to the baseline image. One method ofachieving this is by an imaging device creating a plurality of images.

As noted above, in some embodiments, determining when block facing hasoccurred is achieved by monitoring paraffin on top of the tissue sectionby using some signature of paraffin that differentiates it from tissue.Using this signature, it can be detected when all the paraffin has beenremoved and enough tissue exposed, which would then trigger the “blockis faced” decision. Various methods can be utilized for such detection,and two methods to determine the depth of paraffin on top of the tissueare discussed herein by way of example.

In some embodiments, the block is illuminated with infrared radiationand an image taken. For example, infrared illumination in the 2800-3000cm{circumflex over ( )}⁻¹ wavenumber range (3.3-3.6 mu opticalwavelength range) can be used, as paraffin has a strong absorption inthis range due to stretching modes of the CH bond. If the block isilluminated narrowly in this spectral range (such as by using a quantumcascade type of laser, such as a narrow-band QC laser at 3.28μ), and animage taken with an infrared camera, then the block would first lookblack (when all the block face is paraffin), then a lighter region wouldappear around the portion where tissue is exposed, as the block is cut.

In some embodiments, which is based on the fact that paraffin scatterslight, one (or an array of) sharp light spot(s) is shined onto the blockface. Looking at the diffusely reflected light, the “spot size” shouldcorrelate with the depth of paraffin—thicker paraffin means the lightdiffuses more. For example, if UV illumination is used (with a sharpoptical spot, e.g., using a laser), then it would generate a pointoptical source of fluorescence within the block. The scattering of theUV light on the way in, and the scattering of the fluorescent light onthe way out, would also make the spot broader/more diffuse. Thus, spotsize of the fluorescent radiation would correspond to the thickness ofthe paraffin, thus providing a method for monitoring the depth ofparaffin.

As explained above, hyperspectral imaging can be used by theillumination system. FIGS. 11A and 11B illustrate an exemplary image andits related hyperspectral cube for fatty tissue 230 embedded in paraffin232. The low count of the fatty tissue layer compared to the paraffinshows that it is still below paraffin in the block. The significantlyhigher tissue reflectance in FIGS. 12A and 12B show that it is at thesurface. In this way the layer depth of the tissue can be identified.

As explained above, laser dot diffusion using a diffractive opticalelement can be used for imaging, which can determine depth. For example,FIG. 13 shows the green channel of a tissue block image. Using imageprocessing tools, the gray value of the image can be extracted, as shownin FIG. 14, which illustrates column 2 dots sampled at 1 pixel acrossthe block of the laser dots on the tissue block. Here a single pixelcolumn across the dots has been sampled. When the gray values go belowcertain counts due to tissue absorption, for example, 20 in this case,one can declare that that area of the tissue is faced. The zone betweenpeaks 2 through 4 indicates a faced region in FIG. 14. The gray valuesfrom each row can be combined to obtain a 3D map of tissue facing metricover the surface of the block. In other words, this method could be usedas an absolute measure of facing without progressive images. However,using this method on progressive images improves its efficiency.

The method could include other lighting modalities such as UV or whitelight to determine the boundaries of the tissue in the paraffin block.This boundary can be used as a mask that can be overlaid on the imageobtained using the laser dot array. This narrowed down pixel range helpimproves the accuracy of the facing decision-making algorithm

In some embodiments, the system can be used with an already faced block.N sections at M um thickness are made to complete block facing. After ablock is faced, it can be removed from the microtome and hydrated. Afterhydration it is put back on the microtome. The tissue in the block couldabsorb more moisture than the paraffin and may protrude irregularly fromthe paraffin matrix. It is also very likely that the blade is changed onthe microtome between facing and sectioning. The paraffin block willneed to be registered against the blade after the polish cuts one wouldtake the sections that will be stained and analyzed by the pathologist.

In some embodiments of the automated system, once the block is faced,the automated apparatus will automatically stop facing and start takingsections from the tissue block for transfer to the tape. Thus, theautomated sectioning device will be programmed to take tissue sectionsfrom the block face once it decides that the block is faced, and thistransition occurs without any user intervention or input. In someembodiments, once the automated decision that the block is faced occurs,the automated apparatus will automatically stop facing but user input isrequired to initiate the tissue sectioning/transfer to tape process. Thesystem can include feedback to indicate when the block is fully faced.

In some embodiments, micro-serrations 240 that the blade leaves on theblock face (referred to herein as “blade ballistics”) are evaluated tomake the facing decision, as illustrated in FIG. 15. On the initial cutsections from the sample block, the blade serrations will be only on apart of the block. However, when the block is faced, the serrations willbe over the whole block face. This is because although the block is cutwith a sharp blade, the surface of the block is not flat. On the tissueitself, the serrations are interrupted since the reflection coefficientof the tissue is different than the paraffin block. More specifically,even the best blade has imperfections which leave an imprint on thetissue block during facing (blade ballistics). These marks are going tobe present first on a portion of the block since the block face is notflat and it cannot be in perfect alignment with the blade. Inprogressive images, the blade ballistics are (serrations) left on theblock face on larger and larger areas of the block as deeper cuts aretaken. Stated another way, when facing the block, the whole surface isnot polished at once. As it is polished, the imperfections of the blockappear like fine lines on the block. When the full face of the block isreached, there are micro-serration lines on the whole area of theparaffin block. The reflections of the blade ballistics lines aredifferent on the paraffin and the tissue due to different reflectionconstants. The system compares the regular image of the tissue and theimage captured by the blade ballistics method. This comparison gives aquantitative metric of how much the block is faced and can thereforeidentify whether/when the block is fully faced. Note thesemicro-serrations are not visible under white light but are visible ifilluminated in the UV wavelength so the illumination systems disclosedherein can be utilized with this imaging system of blademicro-serrations.

In some embodiments, consecutive images are taken and compared to aprior image by the device processing the images. In some embodiments,comparing consecutive images to one or more previous images assesseschanges in the colors over time as the block is cut. In someembodiments, size and edges of the tissue and/or the paraffin areevaluated to assess the status. In some embodiments, a qualitativechange in colors of the image is evaluated and quantified. In someembodiments, physical properties of the tissue are determined/evaluatedsuch as a shape, size, edges, outline, etc. to assess the status. Insome embodiments, physical properties of the tissue are extracted fromthe images, the physical properties including a number of pieces oftissue in the sample block and a depth of each piece of tissue.

For full faced blocks, it could be beneficial if not only the tissue,but the entire rectangular paraffin region of the block profile iscaptured. For example, a rhomboid profile may be considered “faced” fortraditional sectioning if sufficient tissue cross section is capturedbut a corner of just paraffin is not. The same is not good for tapetransfer because the tape is applied over the entire block and willcatch on the blade when sectioning in the recessed block corner.Therefore, a determination of where the paraffin is missing would bebeneficial. That is, looking at the paraffin material removed can bebeneficial. This is because in the middle of a cut, if a piece ismissing, the blade is not in contact, so the cut is deeper. Thisviewing/evaluation can be achieved by using a video or series ofpictures as the block gets cut to make the facing decision by viewingthe cut material or trimming as it forms off the blade edge during thecut. Where the trimming forms is where the blade made contact. Lookingat the blade and where the trimming forms, i.e., seeing how the blade isinteracting with the block face at the edge, is akin to doing a linescan of where the blade contacts the block. An image of where verticallyon the block the trimming is formed could be helpful. For example, itcould be difficult to tell from a picture if a previously faced blockhas a region in the middle of the tissue that has since sunken fromdrying out. The tissue may be recessed a few microns around thesurrounding paraffin and retain the gloss sheen of a faced block. Doinga test cut and checking if trimming forms across the entire block iseasier.

There may be instances where tissue is just slightly warped in anotherwise fully faced block. Sections aren't immediately taken afterfacing, rather, after facing, there is usually about a 5-15 minute soakin ice water for hydration that tends to slightly alter, e.g., warp, thepreviously faced block. Usually, a few throwaway polish sections aretaken to reset to the faced block, but the imaging systems can result intaking less waste sections.

In some embodiments, an algorithm can process the images collected ateach section. It will compare it to one or more historical (prior)images and determine if the images are changing such that they indicatea faced block. The system can take progressive images of the block as itis sectioned. In parallel the image processing system will evaluate eachimage and compare it to the historical images from the same tissue blockto decide to continue facing the block or to stop. For example, huevalues could be detected in the initial naive block and as sections arecut, the hue values are compared to the initial values. The algorithmcan subtract the values of consecutive images from the initial value andif exceeds a predetermined value (error function) the system recognizesthe block is faced. Note hue is just one example of the detection basedparameter of the algorithm as other characteristics as noted above canalternatively or additionally form the initial baseline for comparativecalculations/assessments of consecutive images to determine the statusand completion of block facing. In other algorithms, one can use theintensity changes image over image at approximately the same location todetermine facing. On a naive block (unfaced tissue block) due todispersive nature of the paraffin layer on top of the tissue, the imageof the tissue would not be clear and the borders of the tissue would befuzzy. In technical terms the intensity change between the paraffin andtissue border would be gradual. As the tissue is being faced andprogressive images are taken, the paraffin layer over the tissue wouldget thinner until it is totally removed. When one calculates intensityat the paraffin-tissue border in these images, the border would getsharper and sharper. With a suitable threshold value one can determinethe tissue is faced or not.

In some embodiments, people may teach the algorithm when a block isfaced. Thus, in this alternate embodiment, unlike the foregoingembodiments, subjective input by people, e.g., the user, provides theinitial input. In machine learning type of algorithms, progressiveimages taken as more cuts are made need to be annotated by a person toindicate if the facing is achieved or not. This paragraph refers tothese annotations.

In some embodiments, a 2D image or a 3D image can be utilized.

The block can in some embodiments be moved up and down in front of theimaging device. Alternatively, the imaging device(s) can be moved withrespect to the sample block or both the sample block and imaging devicescan be moved relative to one another.

As shown in FIG. 2 and described above, the imaging system takes imagesof the tissue block to assess when facing has occurred. In someembodiments, images are alternatively or in addition taken of the tissueon the blade holder. That is, the imaging system takes images of the cuttissue sections that are lying on the blade holder. The initial cutsections will not have tissue since the naïve tissue block has a layerof paraffin on top of the tissue. As explained above, it takes numerouscuts to remove this paraffin layer by layer to expose the tissue (seeFIGS. 4A-4F). But as the cut sections go deeper into the sample blockand parts of the tissue start to get cut, the contrast increasinglighting, utilizing one or more of the illumination systems describedherein, would enable capture of tissue images of the thin cut sections(ribbon) on the blade holder. That is, under the contrast increasinglighting, the initial sections (containing all or mostly paraffin) don'tfluoresce, but as the portions of the tissue are being cut the systemwill start to detect these tissue portions. When the tissue on theribbon is like the outline of the tissue on the block, the systemrecognizes the block has been faced. The imaging system in thisembodiment would image the tissue block and would have the image of thefull face of the tissue. Comparing this full-face image from the tissueblock and the image collected from the layers removed during the facingoperation on the blade holder, the system would determine how close thefacing of the tissue is achieved so the automated decision of fullfacing can be affected. The system/images can also check that theparaffin parts of the section match the profile of the block forpreventing tape snags. In the automated systems, when it recognized theblock has been faced, in some embodiments, the apparatus canautomatically stop facing, i.e., the microtome can stop cuttingsections.

Regarding light field imaging, in generic terms, a light field cameracaptures the intensity of light in a scene, and also the direction thatthe light rays are traveling in space. This contrasts with aconventional camera, which records only light intensity. One type oflight field camera uses an array of micro-lenses placed in front of anotherwise conventional image sensor to sense intensity, color, anddirectional information. From this super resolution varied spatialinformation, an accurate identification of the “depth” of tissue locatedin the paraffin can be obtained. Various illumination, multi-spectral,hyperspectral, and UV can be utilized to elicit the tissue within theparaffin.

Regarding depth from disparity, one can get depth information from 2Dimages by combining at least two 2D images that are taken from differentpositions with respect to the object. Keeping the camera at a fixedlocation, the paraffin block can be moved in the vertical plane, and orthe horizontal plane to take multiple images. One can then calculate 3Dfeatures of the object based on the 2D images. Image processing toolscan be used to determine the depth information from multiple images ofthe same object. An algorithm could detect key points between stereoimages to calculate disparity. This information can then be used tocalculate the depth information

Regarding depth from focus, another method to identify the depth of aset of tissue embedded in a block of paraffin is using the Imagingsystems optical design to identify the depth of a focused tissuefragment inside of a block of paraffin. UV illuminated Florescentimaging allows for the capture of tissue that is located “inside” aNaïve block of paraffin. Using stepped method of moving the blockforward towards the imaging system a series of images can be obtained ofthe tissue. UV illumination will infiltrate the naïve block causing thetissue to fluoresce. The imaging system has been setup with a shortDepth of Focus (DoF). Then a series of sequential images are captured asthe paraffin block is moved forward toward the camera system in the Zaxis only in steps equal to the DoF where at a given depth thefluorescing tissue will be imaged in focus. Edge detection Algorithmsrun on the green channel (grey scale) images looks to identity the imagewith the most number of “edge” or focused details, thus identifying whenthe majority of the tissue is in focus from the series of displacedimages. This is then correlated to the amount the Block holder movedforward taking into consideration that the depth is modified by theindex of refraction (RI) of the paraffin wax in its solid form. Thisdepth number calculated apriori can then be used to identify how manyslicing cuts on the Block facing Microtome are needed to get to the“faced” condition. Because it doesn't require progressive images, it isa faster method to face the block.

As discussed herein, the illumination systems can include UV, IR, orvisible/broadband for example. Also, in some embodiments, the imagingsystem can capture images of one or more of the illuminated tissue blockface, the cut ribbon on the blade holder, or the cut tissue sectionattached to a supporting medium such as the tape, or a cut tissuesection on a slide. The images can in some embodiments be a video of theblock face or ribbon during sectioning. A computation system processesthe images, for example quantitatively, to determine when the block isfaced. At that point, the microtome cut sections from the sample blockcan be transferred to the tape automatically by the apparatus componentsin the automated tape transfer system for subsequent analysis of thetissue.

The systems herein can also have a linked database to record the datafrom the block facing to allow machine learning improvements.

Comparative and Mechanical Analysis

The present disclosure provides systems and methods for quality controlin histology systems. In some embodiments, a method is provided thatincludes receiving a tissue block comprising a tissue sample embedded inan embedding material, imaging the tissue block to create a firstimaging data of the tissue sample in a tissue section on the tissueblock, removing the tissue section from the tissue block, the tissuesection comprising a part of the tissue sample, imaging the tissuesection to create a second imaging data of the tissue sample in thetissue section, and comparing the first imaging data to the secondimaging data to confirm correspondence in the tissue sample in the firstimaging data and the second imaging data based on one or more qualitycontrol parameters.

In some embodiments, the tissue section is non-conforming if there is nocorrespondence in one or more quality control parameters in the tissuesample in the first imaging data and the second imaging data. In someembodiments, the one or more quality control parameters include one ormore of shape of the tissue sample, size of the tissue sample, or one ormore mechanical damages. In some embodiments, the method can furtherinclude transferring, using a transfer medium, the tissue section to aslide, and the second imaging data comprises an imaging data of thetissue section on the transfer medium or an imaging data of the tissuesection on the slide. In some embodiments, the method can furtherinclude comparing at least two of the first imaging data, the imagingdata of the tissue section on the transfer medium or the imaging data ofthe tissue section on the slide.

In some embodiments, the tissue section is non-conforming if there is nocorrespondence in the shape or the size of the tissue sample in thefirst imaging data and the second imaging data. In some embodiments, theone or more mechanical damages are selected from the group consisting oftearing, shredding, blade marks, wrinkling, cracking, bubbles,insufficient tissue sample, incomplete tissue sample. In someembodiments, the method can further include identifying asnon-confirming a tissue section if one or more mechanical damages arepresent in the tissue sample in the second imaging data but not in thefirst imaging data. In some embodiments, the method can further includeadjusting one or more operating parameters associated with removing ofthe tissue section to correct one or more mechanical damages. In someembodiments, the method can further include approving the tissue sectionif there are no mechanical damages are present in the tissue sample inthe first imaging data and the second imaging data. In some embodiments,the method can further include rejecting the tissue block if one or moremechanical damages are present in both the first imaging data and thesecond imaging data.

After the block facing decision is made, cut tissue sections from thesample block are transferred to tape or (other transport medium) andsubsequently transferred from the tape or other medium to glass slides.The system can ensure one or more of the following: i) the section isnot lost mechanically and remains properly associated with the sampleblock (sample tracking); ii) the section does not suffer mechanicaldamage, such as wrinkling, tearing, cracking, etc. or taken partially toensure it is suitable to work with, e.g., suitable forpathology/histology; iii) the section put on the slide containssufficient amount of tissue and not too much paraffin to ensure it issuitable to work with; and/or iv) the multi-piece tissue in the sampleblock is fully represented on the slide.

An exemplary embodiment of systems and methods for mechanical qualitycontrol of a tissue sample are shown in FIG. 16. Such systems can beconfigured to provide various types/aspects of quality control includingi) a comparative analysis 250 of the section on the slide or transfermedium to the sample block or the section just after cutting from thesample block to ensure there is a proper correspondence (e.g., a checkto ensure the section is not lost mechanically and remains properlyassociated with the sample block from which the section was cut); and/orii) a check to ensure the cut section on the slide or transfer mediumhas not suffered mechanical damage 252, such as wrinkling, tearing,cracking, etc. which could adversely affect pathology; iii) a check toensure the cut section on the slide or transfer medium containssufficient tissue 254 and/or iv) a check to ensure the multi-piecetissue of the sample block is fully represented on the slides 256. Thesemultiple aspects can be used alone or in combination with one of theother aspects or in combination with two or more of the other aspects.

Various quality control analyses can be performed on the tissue samplesduring microtomy, including but not limited:

-   -   i) a comparative analysis of the section on the slide or        transfer medium to the sample block or the section just after        cutting from the sample block to ensure there is a proper        correspondence (e.g., a check to ensure the section is not lost        mechanically and remains properly associated with the sample        block from which the section was cut); and/or    -   ii) a check to ensure the cut section on the slide or transfer        medium has not suffered mechanical damage, such as wrinkling,        tearing, cracking, etc. which could adversely affect pathology;    -   iii) a check to ensure the cut section on the slide or transfer        medium contains sufficient tissue; and/or    -   iv) a check to ensure the multi-piece tissue of the sample block        is fully represented on the slides.        These various quality control analyses can be used alone or in        any combination thereof.

In some embodiments, a system is provided to check the condition of thetissue on the microscope slide. This provides another aspect of qualitycontrol to ensure that the tissue on the slide is in the propercondition for further analysis/evaluation.

In some embodiments, a system is provided to check if the section on theslide contains a sufficient amount of tissue to render it adequate foranalysis/evaluation. That is, the system ensures that not too muchembedding material, e.g., paraffin, is in the section on the slide. Thisprovides another aspect of quality control to ensure that the tissue onthe slide is in the proper condition for further analysis/evaluation.

In some embodiments, a system is provided to check tissue on the tape(or other transport medium) as an interim quality control check, e.g.for tracking purposes or for tissue integrity purposes (in propercondition for analysis).

In some embodiments, a system is provided to check that a multi-piecetissue in the sample block is fully represented on the slide to ensure apiece is not missing. Such missing piece could require the pathologistto order recuts. This integrity check provides another aspect of qualitycontrol.

In some embodiments, the tissue comparison can be facilitated by anillumination subsystem and imaging subsystem. In some embodiments, itcan be provided in an automated transfer system, described in moredetail below. Thus, within an automated sectioning and cut tissuetransferring apparatus, an image based automated tissue comparisonsystem is provided—a machine vision system for automating tracking andquality control of the tissue section. The systems can be used tocompare the tissue outline on the glass slide to the tissue shape on theblock face to ensure proper identification of the slides. In someembodiments, the image of the cut tissue section is taken on a transportmedium which via a controller transports the cut tissue section awayfrom the sample block. In some embodiments, the step of comparingcomprises the step of determining an outline of the tissue on the sampleblock and an outline of the first cut tissue section. In these systems,the image can be taken of the block face just prior to taking the tissuesection (or alternatively or in addition taken of the slice after cutfrom the sample block) and the image is used as a comparison image for asubsequent image of the same section on the slide. A computational stepcompares the two images and ensures that there has not been asignificant change in one or more parameters or features. If asignificant change is detected, for example if the change exceeds apredetermined parameter in some embodiments, it can be provided as afeedback signal for corrective action. The illumination system aids indiscernment/differentiation of the tissue and paraffin which can beimaged by the imaging system for evaluation. Thus, in this system andmethod, the differentiation of the tissue from the paraffin is enhanced,and the images are taken, on the block, transfer medium, slide and/orother locations. For example, the step of comparing the image includesevaluating and quantifying a change in colors of the image. In someembodiments, the step of comparing the image includes determining if achange exists in one or more of location, spatial shape and integrity.In some embodiments, a qualitative change in colors, granularity, etc.of the image is evaluated and quantified. In some embodiments, grayscale imaging can be used. Thus, if the change exceeds a predeterminedparameter, a feedback signal is provided for corrective action Variousillumination systems and imaging systems are discussed above.

A system and method of computational processing after the visualizationsystem utilizing optics as disclosed herein is operated can also beprovided. Thus, the visualization system utilizes appropriate optics(illumination system/method, imaging system/method, detectionsystem/method) to be followed subsequently by computational processing.This computational processing provides comparative assessment of imagesto determine tracking or other aspects of quality control.

The illumination system enhances visual/imaging differentiation of thetissue and paraffin and the imaging systems takes images of theilluminated tissue/paraffin for subsequent comparison. The illuminationsystem enhances visual/imaging of the tissue on the block face and theimaging systems takes images of the illuminated tissue for subsequentcomparison for quality control. Such imaging of the individual tissuesection is done prior to separation of the cut tissue from the block orotherwise prior to transfer to the slide, to provide a base forcomparison. Subsequently, such assessment of the individual tissuesections is done after the cut tissue section is transferred to theslide. Various ways to differentiate are described below. Variousimaging systems and various locations for imaging are also discussedbelow. Also discussed in detail below are various embodiments ofillumination systems which create and/or enhance the contrast betweenthe tissue and paraffin, relying on the properties of the paraffin andthe tissue. In some embodiments, the tissue of the sample block isembedded in embedding medium and the method further comprises the stepof one or both of i) illuminating the sample block with wavelengths oflight to increase a contrast between the tissue of the sample block andthe embedding medium of the sample block in which the tissue isembedded; and ii) illuminating the first slide and or transport mediumcontaining the first tissue section with wavelengths of light toincrease a contrast between the tissue and the embedding medium in whichthe tissue is embedded.

Comparative Analysis (Block Versus Section)

There are various ways to achieve a comparison to determine if thetissue section on the slide or in transport between the tissue block andthe slide is within pre-set criteria so as to correspond to the samepiece of tissue on the sample block. The system can, in addition oralternatively, compare the tissue section on the microscope slide to thetissue section that had just been cut from the sample block to determineif within a pre-set criteria they correspond to the same tissue samplecompare. The tissue section on the transfer medium can additionally oralternatively be compared to sample block or the tissue on themicroscope slide. The decision system can examine one or more parameters(characteristics/features) to make the comparison. A decision-makingalgorithm of the decision system then provides a cue or what if anyaction to take. These quality control systems speed the process, improvethe process via fewer errors, and result in fewer wasted sections. Insome embodiments, the comparison determines if there are matchingidentifications between the tissue block and the tissue section, and caninclude matching tissue contours and edges, and bar codes on the sampleblock 260 and on the slides 262, as shown for example in FIGS. 17A and17B. In some embodiments, the comparison determines a match between thetissue block and the tissue section in transport, as shown in FIGS. 18and 19, which illustrate exemplary images taken of the tissue section intransport. Below are some examples, it being understood that othercriteria can be used utilizing for example, light, composition,mechanical characteristics, etc.

It will be understood that instead of comparison of the tissue on theslide to the block face, the comparison can be made of the tissuesection on the slide to the cut tissue section (slice) after cut fromthe sample block. Thus, the discussion of the systems for assessing theblock face, including the illumination and imaging systems, which arediscussed herein are fully applicable for assessing the cut slice forcomparison to the tissue section on the slide.

The block facing decision can in some embodiments be determined inaccordance with the inventive concepts in commonly assigned provisionalapplication filed on the same day as the present application andentitled “Systems and Methods for Assessment of Tissue Block Facing inAutomated Tissue Transfer Systems,” the entire contents of which areincorporated herein by reference. Other methods can also be utilized.Once the block is faced, the block is ready for cutting thin tissuesections for transfer to a transport medium (e.g., tape) and then toslides analysis. Thus, quality control systems/methods disclosed hereincan be utilized with such block facing decision. In some embodiments,automated methods (processes) and systems can be used to automaticallyface the tissue in the paraffin block via a fully automated tissuesectioning device wherein once faced, the tissue is automatically cutfrom the block face, automatically transferred to tape and the tape isautomatically moved via rollers to advance the cut tissue and positionsubsequent portions of the tape over the block face for subsequenttransfer of cut tissue sections to the tape. In some embodiments, theautomated tissue sectioning apparatus also includes a slide station andthe tissue sections held on the tape are automatically transported toand transferred in the automated apparatus to glass slides for analysis.

Images can be taken of the cut tissue section on the tape (or othertransfer medium) after it is cut from the block and adhered to the tape.Images can additionally or alternatively be taken of the cut tissuesection after it has been transferred from the tape to the slide. Analgorithm will process the images collected at each section with acomputational step to compare the image on the tape and/or slide to oneor more historical (prior) images and determine if there is asignificant change in characteristics such as location, spatial shapeand integrity. The location of the tissue sections on a (glassmicroscope) slide. The tissue section has a rectangular shape and thelarger ones are approximately 28 mm×22 mm. The usable area of the glassslide is approximately 50 mm×25 mm, the tissue section can betransferred anywhere on the slide, including rotational changes. Butsuch a haphazard transfer is not preferred. Mechatronic systems canensure that the similar sized tissue sections are deposited at similarlocations on the glass slide. In addition, the QC system will check ifthe transferred tissue in the expected location and orientation. Spatialshape can be considered as part of the previous explanation. Tissueintegrity refers to not having defects such as bubbles under the tissue,tears, blade marks, shredding, cracks, and missing pieces. Note thehistorical (reference) image can include an image on the sample blockbefore it is cut and/or an image of the section (slice) after it is cutbefore it is placed on the tape or slide. Thus, the system will takeprogressive images of the block as it is sectioned. The system will alsotake images of the cut tissue section on the tape and/or on the slide.In parallel, the image processing system will evaluate each image andcompare it to the historical images from the same tissue block to ensureit has not changed. For example, hue values could be detected in thesample block and as sections are cut, the hue values are compared to theinitial values. The algorithm can subtract the values of consecutiveimages from the initial value to assess if it is a match. Note hue isjust one example of the detection-based parameter of the algorithm asother characteristics as noted herein can alternatively or additionallyform the initial baseline for comparative calculations/assessments ofconsecutive images to determine matching. One can use the intensitychanges image over image at approximately the same location to determinefacing. On a naive block (unfaced tissue block) due to dispersive natureof the paraffin layer on top of the tissue, the image of the tissuewould not be clear, and the borders of the tissue would be fuzzy. Intechnical terms the intensity change between the paraffin and tissueborder would be gradual. As the tissue is being faced and progressiveimages are taken, the paraffin layer over the tissue would get thinneruntil it is totally removed. When one calculates intensity at theparaffin-tissue border in these images, the border would get sharper andsharper. With a suitable threshold value one can determine the tissue isfaced or not.

In some embodiments, the quality control is not a machine learningalgorithm trained by people. Thus, in some embodiments, the methodologywill not depend on expert humans teaching a machine learning algorithmhow to assess quality or a match. Note though in some implementations,the results of the classical image processing methods could be fed intoa machine learning algorithm to train it with the expectation that themachine learning algorithm can handle more generic cases. This methodwould cost effectively increase the number of annotated images used forAI algorithm teaching phase. In machine learning type of algorithms,progressive images taken as more cuts are made need to be annotated by aperson to indicate if the facing is achieved or not. But one canalternatively use classical image processing techniques to annotate theimages that are easier for such algorithms (more structured) and trainan AI algorithm to extend the applicability of the overall algorithm tomore general (unstructured) data.

In some embodiments, the illumination system enhances the detection oftissue. Since in some cases processed and embedded tissue has a veryfaint color compared to the paraffin matrix surrounding it, it isdifficult to reliably capture the tissue outline with regular imaging.Thus, applications of certain ranges of wavelengths can increase thecontrast of the tissue against the paraffin. For example, UV light isused to increase contrast ranging from 320 nm to 400 nm. Such detectionenhancement facilitates comparison of the tissue on slides (or tape) tothe block. It also facilitates checking the suitability of the tissuesection on the slide (or tape) in accordance with other quality controlaspects.

In some embodiments, increasing of the contrast of the tissue fromparaffin is achieved using multispectral images, i.e., varyingwavelength radiation, to illuminate the tissue block at the sameinstance of the block section. These images are combined to increase thecontrast of the tissue section in the tissue block. These highercontrast images are easier to compare.

In some embodiments, UV radiation can be used to illuminate the tissuesince tissue samples fluoresce when illuminated by UV radiation. UVradiation as used herein can be interpreted broadly as wavelengthsshorter than optically visible blue light. Note, however, in someembodiments, the actual wavelength range available may include some ofthe blue end of the light spectrum. The tissue sample glows under UVillumination (e.g., can glow green) with a diode since biological tissuehas numerous fluorescent molecules that are relevant in the presentcontext, including NADH, FADH. Paraffin, however, does not fluoresceunder the same condition, and the paraffin block scatters the visiblepart of the UV light source and can appear a different shade or color,e.g., can appear bluish. Three advantages of UV light can beappreciated. First, the UV light penetrates into the paraffin block, andthe fluorescent radiation escapes from the paraffin block, so that themethod/process can clearly visualize tissue samples buried within theblock. This is in contrast with looking at the block under visiblelight, which is strongly scattered by the paraffin wax, and the buriedtissue samples are not clearly visible and may even be invisible.Second, the color (hue) of the emitted light (more precisely, thewavelength ranges of the fluorescent radiation and the passivelyscattered radiation) provides a clear contrast between the tissue andthe paraffin wax, thus permitting straightforward detection andsegmentation of the tissue samples. Third, by imaging the cut section onthe tape (or other transport medium) and/or the slide, and by observingthe distinct fluorescent radiation being emitted by the tissue, it ispossible to precisely detect the tissue placement for checking integrityand checking that it matches the pre-cut tissue section.

In some embodiments, another range of illuminating wavelengths utilizedto illuminate the block face and the cut tissue section on the tapeand/or slide lies in the infra-red range. Paraffin wax has acharacteristic infrared absorption spectrum. If reflection spectrum isacquired from the face of the paraffin block in an imaging mode, byselecting the tissue portion from processing the UV image (or if thereflection IR spectroscopy is performed in imaging mode), the tissue canbe detected when the IR spectral signature of paraffin is diminished ina localized manner over the tissue sample.

Infrared spectroscopic methods can be used in some embodiments to detectthe hydration state of the tissue in the block, utilizing thecharacteristic IR absorption spectrum of liquid water.

One method is shown in the flow chart of FIG. 20 utilizing the UV orinfrared tissue illumination. The steps can include i) illumination by apre-selected range of wavelengths in step 270 (e.g., the UV range); ii)using appropriate optics to create images on a color camera, i.e., acamera that simultaneously takes images in multiple wavelength rangessuch as an RGB camera for example, of the block face (step 272) and thetape and slide (step 274); iii) using color and intensity informationfrom the resulting image to segment out the portions where tissueresides (step 276); iv) monitoring the size of the tissue region, andthe edges of the tissue region (step 278); and v) comparison of imagesto detect conformity/changes (step 280). Optionally, a comparison canalso be done between the images and the baseline image (step 282). Inparticular, as explained above, the outline of the tissue sample can becompared to the expected outline of the tissue sample from the baselineimage, and the comparison of the actual outline to the expected outlinecan confirm the source of the tissue sample, that is, the tissue samplecomes from the tissue block having a barcode associated with the slide.

This information is used to compare the tissue on tape to the image ofthe block and tissue on tape image to image of the block. These firstcomparison (tape to block) enables identification of tissue pick up fromthe block face with the transfer medium (tape). The tissue could bepicked up partially, or part of the tissue could be torn and rotated. Ifthe tissue on tape is not high quality enough then it is not worthtransferring to the glass slide. This saves time and resources. Thesecond comparison (slide to block). The tissue transferred to the glassslide may have bubbles, tears, missing pieces etc. these glass slidesshould not be given to the end user as final product as they will besub-par or useless. The algorithms may result in a recut of the sectionor warn the user about a bad block. A bad block could have a tissue thatis not embedded properly in the lab before it is introduced to thesystem.

As discussed above, in the automated apparatus, these steps areautomated and do not require user input during the process. Note thatintensity in a gray scale can be used as alternative to color images.Thus, in this method, UV radiation or other wavelengths enhance thetissue/paraffin contrast as discussed herein, and observations are madeof both the block face and the cut tissue section. In this method,appearance of tissue-fluorescence in a cut tissue section is detected.This can be done either based on the total fluorescence from the ribbon(so that there is no need to fully image the ribbon face-on) or,alternatively, the ribbon could be imaged face on. Such imaging is doneon the cut tissue section (on the tape or slide), and comparing theimage of the tissue section on the tape or slide with the image of theblock face (each with UV illumination). The images are processed andevaluated for tissue segmentation (or other characteristics/parametersdescribed herein) as the size and edges of the tissue regions can beassessed and quantified to a match.

In some embodiments, multi-spectral illumination can be used, andinvolves spectroscopy, i.e. acquiring information from multiplewavelengths or colors, e.g., a Fourier-Transform Infrared Spectroscopecan be employed. This can be done in an imaging mode or by simplespectroscopy from a spot that is chosen by some other way (preliminaryUV imaging) to focus on top of the tissue. In either case, the goal isto employ the distinctive infrared spectral signatures of paraffin andtissue in order to (i) determine if there is paraffin on top of thetissue, or if the paraffin has been removed and the tissue exposed; or(ii) imaging the ribbon on the tape to detect presence of tissue andreduction in the amount of paraffin; or (iii) imaging the ribbon off thetape and on the slide, to detect presence of tissue and reduction in theamount of paraffin.

In some embodiments, visible/broadband illumination is used to image theblock face and/or the tissue section on the tape and/or on the slide forcomparison to detect (i) a qualitative change in the image, such as thetissue portion becoming more visible/darker brown/with better definededges on the block, or ii) by looking at the cut tissue section anddetecting tissue by using color/intensity information.

It should be appreciated that the above methods can be combined so thatmore than one illumination system can be utilized.

In the methods herein, used individually or in combination, there is acomputational step as discussed herein where the multi-color image orspectrum is subjected to analysis, and an appropriate change detected inorder to ensure the cut tissue section remains properly associated withthe sample block. This computational step can include image comparisonsto an original or baseline image, or to prior images, and the comparisonof hue, intensity, boundaries, etc. can be quantified for calculation ofthe extent of differences between the images for assessment.

In the case of tissue imaging on the tape or on the slide, the referenceimage could be the image of the tissue in the block.

The automated system in some embodiments can capture variousconsequential variables like speed, temperature, humidity, time outs,etc. for input to machine learning to improve the process. If the QCsystem deems sections as good quality and this trend correlates with acertain range of operating temperatures this can be the bases of amachine learning algorithm to make sure all future blocks with similartissue are cut under conditions that result in good quality cuts.

The decision-making process can determine that there is not a propercorrespondence (comment criteria) between the tissue on the slide andthe tissue on the sample block. The decision-making process can alsoinclude a finding of a proper correspondence between the tissue on theslide and the sample block, but a finding that the tissue is distortedor damaged and therefore not usable.

Note the decision-making algorithm provides a cue of recommended actionand following the recommendation can be optional. However, in someembodiments, whether or not the cue is taken, the information isrecorded in a database which potentially can use machine learning toimprove the decision-making algorithm.

In some embodiments, the system can provide a cue when to select anothersection to transfer to a slide. For example, selection can be every nthsection or when the image of successive sections changes by somefractional amount of criteria.

An example of an inaccurate match (correspondence) will now bedescribed. As discussed herein, the biological tissue is embedded in aparaffin matrix (or other embedding medium) forming the sample block.However, the tissue embedded in paraffin blocks may not always have highcontrast compared to the paraffin matrix. This can adversely affectimage analysis as it could be more difficult to differentiate the tissueand paraffin, thus making it more difficult to assess the shape, e.g.,outline, of the tissue on the sample block. Thus, the illumination andimaging systems disclosed herein provide a system and method to improvedifferentiation of the tissue and paraffin so the images of the tissue,and thus the determination of the tissue shape, e.g., outline, can bedetermined (analyzed) with more accuracy. This ensures a more accuratecomparison of the tissue by reducing the potential distortion of theimage, e.g., by inaccurately including the paraffin as part of thetissue image. In other words, if the “base” or “input” image, defined asthe initial image which is intended to define the tissue contour on thesample block for later comparison to the tissue contour on the slide,includes paraffin, and the “second” or “output” image, defined as thesubsequent image of the tissue on the slide, is processed without theparaffin, then a false determination of a non-match could occur.Similarly, if the base image does not include paraffin, but the secondimage of the tissue on the slide is defined with the paraffin, then afalse determination of a non-match could occur. Conversely, if notaccurately differentiated and processed, false matches could also occur.

The section thickness ranges from about 1 to about 15 um thick but mostcommonly are about 4 um thick. At this thickness, the contrast ratiobetween the paraffin matrix and the tissue section is very low. In someembodiments, to ensure the quality control system is accuratelycomparing the tissue itself or accurately recognizing the tissue and theparaffin on the images, the contrast ratio between the tissue andparaffin is enhanced by the systems and methods disclosed herein. Theimage post-processing efficiency increases when the image has highcontrast ratios as explained above. Tissue starts to fluoresce whenilluminated with light of a certain wavelength range and the paraffindoesn't fluoresce at the same wavelength range. This creates a highcontrast image. Therefore, the sample block is illuminated with a rangeof wavelengths of light to increase the contrast ratio between thebiological tissue and the paraffin matrix. Similarly, the glass slidescontaining the tissue sections deposited thereon are illuminated with arange of wavelengths of light to increase the contrast between thebiological tissue and the paraffin matrix. The slide with the tissue canbe back illuminated or alternatively illuminated from the front, withback and front of the slide defined based on where the camera is placedwith respect to the glass slide. Thus, in these embodiments, the systemcan better differentiate the tissue from the paraffin for comparativeanalysis of the sample block and slide.

In some embodiments, the wavelength of the light can be controlled withfilters or LEDs with a given range of wavelength emission. In someembodiments, on the image capture side, filters could be provided toenhance the image capture. The set of lights sources, filters and thecamera can be referred to as the imaging hardware.

Mechanical Property Analysis

In the illumination and imaging systems disclosed herein, the systemscan be utilized to ensure the cut tissue section does not suffermechanical damage, such as wrinkling, tearing, cracking, etc. whentransferred to the slide. Such systems can also be utilized to ensuresufficient amount of tissue is on the slide. In comparing the tissue ontape or slide to block face if there are significant differences insurface area then the algorithm can say there is not sufficient tissueon the slide.

Composition can be measured by sampling vapor above the samples or byextracting material from the samples by bombardment and can be detectedby mass spectrometry, vapor phase chromatography or other methods. Thisin the context of mass spectrometry, in the sense that detecting thesubstances in the vapor over a substance.

Mechanical properties can be measured by vibration, atomic forcemicroscopy or other methods. One can determine material properties usingvibrations in this case it is a stretch but included here forcompleteness. In the case of atomic force microscopy the attractiveforces on the probe and the material being probed can be used as anindication of material properties. The reason this is included here isfor completeness of facing decision sensors. It is not an imagingmodality.

In some embodiments, the quality control system can check for sampleorientation and/or inversion. In some embodiments, the quality controlsystem determines tissue orientation variations on one or both of thetransfer medium and the slide to alert the user if components of theautomated apparatus need adjustment.

Sample Sufficiency Analysis

In some embodiments, the system can perform a check to ensure the cutsection on the slide or transfer medium contains sufficient tissue. Forexample, the system would compare the surface area of the tissue inblock face and the section on the glass slide. If the two areas aresimilar in a pre-determined range then the tissue integrity ispreserved. Another example is the existence of bubbles under thesection. A comparison of intensities of the two images would reveal ifbubbles exist or not. It could also provide where the bubbles are. Ifthe bubbles only exist in the paraffin matrix then it is not a criticalfailure. On the other hand, if the bubbles are on the tissue then thisindicates a low quality tissue on slide.

Tissue Sample Completeness

In some embodiments, the system can perform a check to ensure themulti-piece tissue of the sample block is fully represented on theslides. In some tissue blocks there may be multiple pieces of tissue.The histotech places the tissue pieces in the plastic cassette and whiletrying to push them to the bottom of the cassette they pour warmparaffin wax. In certain cases some of these tissues move and are notplanar with the rest. During sectioning one way to confirm is to comparethe block face image to the tissue on tape or glass slide. If they havethe same number of tissues then the overall tissue integrity ispreserved.

Tracking and Printing

In some embodiments, a just-in-time glass slide label printing protocolcan be implemented where the labels for the slides are printed after thetissue samples are cut from the tissue block. In this manner, the glassslide is barcoded or labeled by a just-in-time printer with the barcodederived from the block that was just sectioned at the microtome, suchthat the immediately cut tissue section is then placed on the newlyprinted barcoded slide. In some embodiments, the next tissue section iscut and label printed only after the preceding tissue section has beenplaced on the slide and labeled, and optionally confirmed to beassociated with the tissue block. In some embodiments, real-time updatesare communicated via the laboratory information management system todevice software, while also enabling real-time tape marking of thebarcode data.

In some embodiments, the present disclosure closes the loop of trackinga cut tissue sample while it is in transit between the block face andthe destination of the glass microscope slide. providing, therein,real-time, updatable tracking and identification of the tissue sectionlocation in the tissue processing device vis-a-vis the LIMS data. Insome embodiments, the transfer system is labeled to associate the tissuesections placed on the transfer system with the tissue block from whichthe tissue sample is cut. In some embodiments, the scanned barcode datais tracked from the point that the section is placed on tape, whichincludes replicating the information by a tape marking/printingmechanism; replicating the barcode data by just-in-time digital printingof the label on the glass slide; transferring the tissue to the glassslide; scanning the printed barcode on the glass slide; and thenverifying exact correspondence among the barcode data on the block, theprinted tape, and the printed slide; and optionally, communicating asummary report to the LIMS.

The present disclosure is directed to tracking of tissue sections cutfrom a sample block and just-in-time printing of the glass slideidentification label of cut tissue sections in an automated tissuetransfer apparatus. The information from the tissue block is transferredto the slides in real time and ensures an accurate 1 to 1 tracking andlabeling of the tissue sections. The present disclosure overcomes theproblems and deficiencies of the prior art by the implementation of ajust-in-time glass slide label printing protocol. In particular, one ormore tissue sections from the same tissue block are cut before the glassslide is actually printed. The glass slide can then be barcoded orlabeled by a just-in-time digital printer with the barcode derived fromthe tissue block that was just sectioned at the microtome. Theimmediately cut tissue section is then placed on the newly printedbarcoded slide.

Tracking and identification of tissue sections can be achieved by anumber of integrated sub-assemblies and mechanisms, including, by way ofa non-limiting example, a tape marking or a tape printing device thatreplicates the barcode data associated with the incoming tissue block,which is captured by a barcode reader, integrated within the automatedtissue sectioning machine. The barcode data, generated by laboratoryinformation management system (LIMS) software, is embodied by theprinted adhesive label, attached thereon to the plastic cassette holdingthe tissue block. In some embodiments, the tissue transfer medium mayhave location markings or barcodes printed on it before it is used fortissue transfer. When the tissue transfer medium is an adhesive tape,the location markings can be placed on the tape during a tape conversionoperation.

Typically, the tissue sample (also referred to as tissue block or sampleblock) is provided in a plastic cassette and is embedded in paraffin waxor a similar material. The plastic cassette provides the features forthe sample block to be held in a microtome clamp. Once the sample blockis secured in the microtome clamp for cutting (sectioning), the newblock is first subjected to sectioning with relatively thick sections toremove the 0.1 mm-1 mm layer of paraffin wax on top of the tissue sampleto expose the tissue sample. After removal of this superficial paraffinlayer and when the complete outline of the tissue sample is exposed,then the block is ready to be sectioned. This process for removing thisparaffin layer and exposing the large cross section of the tissue isreferred to as block facing. Once this paraffin layer is removed, inclinical and research settings, the tissue is typically sectioned to 3μm to 5 μm thickness. That is, when enough paraffin has been removed(the block is referred to as “faced”), subsequent block sectioningprovides tissue sections for placement on glass slides for analysis(processed further for evaluation). The tissue sections cut from thesample block can be transferred to slides, such as, for example, using atape transfer mechanism. In some embodiments, the process can beautomated as, for example, disclosed in commonly assigned U.S.Publication No. 2017/0205317. Other examples of an automated apparatus,and variations thereof are disclosed in U.S. Publication No.2017/0003309 and U.S. Publication No. 2017/0328818. The entire contentsof these three publications are incorporated herein by reference. It isunderstood that the automated tissue apparatus provide examples ofautomated apparatus as the illumination/imaging systems and the qualitycontrol systems can be used with other automated apparatus. Also, asdiscussed herein, the section tracking systems can be used with manualsystems and methods.

In reference to FIGS. 17A and 17B, incoming tissue blocks include alabel attached to the plastic cassette a barcode number. The barcodedata, generated by laboratory information management system (LIMS)software, provides information about the source of the tissue samples,for example, the barcode information includes an accession number andblock ID. In some embodiments, this information can also include thepatient name and date when the specimen was obtained. Depending on thelab, additional information may be included. In addition to the barcode,the label or the etching on the block could include human readablealpha-numeric version of the data. In some embodiments, the microtomydevices may be in communication with the LIMS enabling real-time LIMSupdates to be correctly matched to the proper tissue block from theinitial pick-up by a robotic arm right through to actual tissuesectioning and delivering the tissue sections to slides. The barcodeinformation on the tissue block, optionally updated, is also printed onthe slides such that there is a 1:1 correspondence between the tissuesections from the tissue block and the slides. As is described in moredetail below, the labels for the slides can be printed after the tissuesections are cut from the tissue block.

In reference to FIG. 21A, a barcode reader is provided to scan thebarcode associated with the incoming tissue block. The scanning can takeplace at the point of tissue sectioning. The barcode information is usedto interrogate the data from the LIMS to determine the number ofsections that needs to be cut, thickness of the sections and otherprocessing parameters. Next, one or more tissue samples can be cut bythe microtome and are transferred to the slides that are also labeledwith the barcode data associated with the tissue block to create a 1:1association of the tissue block meta data with the tissue sections onthe slides. In some embodiments, the slides are labeled based on thetissue block barcode with iterative variations. For example, if theblock barcode is 12345, the first slide barcode could be 12345-a and thesecond one could be 12345-b and so on. The slide labels are printed justin time before tissue transfer to the slide.

In some embodiments, the tissue sections are transferred to the slidesusing a tape. It should be noted that transfer medium (also referred toas transport medium) other than tape can be utilized. Therefore,references to tape herein are used for convenience as the systems andmethods disclosed herein are fully applicable to other transfer mediumnot just tape.

In some embodiments, the tape transfer system is configured to enablethe tracking and identification system. The tape transfer system can bemarked with information that can be associated with the tissue block.Such markings can be done after the tissue block is received in thedevice or be pre-printed on the tape. In some embodiments, the transfermedium (tape) may include location markings. The lab assigned block IDcould then be associated with this location marking at the point thatthe tissue section is picked up by the transfer medium, the two IDscould be associated by means of a software tool. It is also possiblethat there is no physical marking on the tape, but a virtual marking isinferred based on a position encoder in the device, which keeps track ofabsolute position on the tape and is able to associate this absoluteposition, explicitly or implicitly via a suitable control algorithm, tothe section in question.

FIG. 21A is a schematic view of some embodiments of a device with anautomated tape transfer apparatus (system) 300. In some embodiments, thetape transfer system is configured to enable the tracking andidentification system. FIG. 21A illustrates the path of the tape 302 fortransporting cut tissue sections after the block is fully faced. FIG.21A shows a microtome 304 that is used to hold the sample blocks and cutthe sections. The microtome 304 holds a sample block comprising a tissuesample that is enclosed in a supporting block of embedding material suchas paraffin wax. The microtome 304 includes a blade (not shown) alignedfor cutting slices (or sections) from the face of the tissue block. Oncethe tissue samples are cut from the tissue block, they are mounted onthe tape to be transported to the slides.

The automated microtomy device can also include a tape marking system306, in communication with a barcode scanning reading system 308. Thetape marking system 306 can be used to mark the adhesive tape with thebarcode information captured from the label attached to the plasticcassette holding the tissue block. A variety of printing methods,including thermal or continuous inkjet printing technologies may be usedwithin the automated tissue sectioning device, for this purpose. Inaddition, thermal transfer print units may also be used, in order togenerate identifying information on the tape that tracks the incomingtissue block to the cut section on tape, in situ, wherein thisinformation then enables correspondence between the section on tape andthe section on slide, ensuring that the integrity of the sample ismaintained.

The tape transports the tissue sections from the sectioning microtome304 to the slide station 310. In some embodiments, the device can alsoinclude a glass slide printing system 312 and a barcode reading system314. The printing system 312 prints the labels for the slides thatassociate the tissue sections to be placed on the slides with the tissueblock from which the tissues are cut. In some embodiments, the automatedsystems can associate the barcode identifier on a tissue block with themarkings on the tape transfer medium and then associates the markings onthe transfer medium with the barcode printed on demand on the glassslide. This is in the context of a fully automated tissue sectioningdevice and provides just-in-time printing of real-time LIMS data ontothe glass slide. It should be noted that other transport devices/systemscan be used. The tissue in/on these other transport systems can betracked in accordance with the tape printing systems described herein.Therefore, the systems, e.g., tape printing systems, slide printingsystems, etc. described herein are fully applicable to the section(slice) on the various transport systems. Due to the tracking of thetissue at different stages in the apparatus, e.g., block to new slice,slice on transport, slice on slide, multiple levels of tracking areprovided. In some embodiments, the label may link a slide to relevantLIMS-based information, such as the originating sample tissue block andthe sectioning date. Tissue blocks may be similarly labeled. Toaccommodate pre-labeled blocks, an optical reader, such as a barcodereader may be used to read the block label to generate the associatedslide labels.

In some embodiments, the printhead of the tape marking device may beplaced in the tape path, at some arbitrary point before tissue transferfrom the tissue block to the tissue transfer medium. Wherein a barcodereader, adjacent to the block plastic cassette, at the point ofsectioning, reads the barcode data on the plastic cassette, such thatthe barcode data or other alphanumeric data is replicated on the tapetransfer medium. In some embodiments the marking on the transfer mediummay not be a replica of the barcode on the block cassette but the twomarkings may be associated through a software structure. In someembodiments, the information printed/etched on the tape can enabletissue tracking inside the microtomy device. The match between theblock-barcode, and tape-mark (internal to the device) can be ensured bythe mechanical operation of the device, or by scanning the sectiontogether with the tape mark, for example, using a camera. The glassslide can be printed with a barcode related to the barcode on the tissuecassette barcode, and then finally, scanned, thereby ensuring 1-1mapping or correspondence between the barcode data associated with theincoming block and the barcode data printed on the glass slide label,thus ensuring tissue sample tracking, a critical aspect of regulatoryquality assurance.

In some embodiments, a match between the tissue in the block, and thetissue section on the slide can be confirmed based on camera images andimage processing. In some embodiments, an imaging or scanning device canbe employed to check that the label on the slide was printed correctlyso that the tissue section on the slide is associated with the correcttissue block. At each point, the physical replication or printing of themarkings on the tissue carrying receptacles (block cassette, transfermedium, or the glass slide) are recorded and cross-checked for correcttracking of the tissue. This provides a situational awareness of howtissue and cut tissue sections traverse through the tissue processingapparatus. In some embodiment, the presently disclosed methods andsystems can utilize a quality control imaging system as disclosed, forexample, in co-pending U.S. Application No. 62/980,203, filed on Feb.22, 2020, which is incorporated herein by reference in its entirety.

The scanned barcode data is tracked from the point that the section isplaced on tape, which includes replicating the information by a tapemarking/printing mechanism; replicating the barcode data by just-in-timeprinting of the label on the glass slide; transferring the tissue to theglass slide; scanning the printed barcode on the glass slide; and thenverifying exact correspondence among the blockface; tape; and slidebarcode data; and optionally, communicating a summary report to theLIMS.

In some embodiments, the present disclosure also provides a system tocheck tissue on the tape (or other transport medium) as an interimquality control check, e.g., for tracking purposes or for tissueintegrity purposes (in proper condition for analysis).

In reference to FIG. 21B, in some embodiments, the tape can bepre-printed. The transfer medium (tape) may be pre-printed with locationmarkings on it by a conversion process outside the device. The labassigned block ID could then be associated with this location marking atthe point that the tissue section is picked up by the transfer medium,the two IDs could be associated by means of a software tool, such asmaking corresponding entries in a database table.

With reference to the tracking flow chart of FIG. 22, the barcodeassociated with the incoming tissue block is scanned (step 320) at thepoint of tissue sectioning, using an integrated barcode reader. In someembodiments, in order to replicate the LIMS-based barcode dataassociated with the incoming tissue block, the section transfer system(such as tape) is marked with identifying information (step 322), whichmay be the same as the barcode or include some arbitrary rendition ofthe LIMS data. Additionally or alternatively, the physical location ofthe tissue section on tape is kept track of, by keeping track of thelength of tape from a fiducial mark, such as the beginning of the tape.Next the tissue sections are cut from the tissue block and are placed onthe tape in association with the markings on the tape (steps 324 and326), which also correspond to the barcode information on the tissueblock. Once the tissue samples are cut from the block, the glass slideis labeled by a just-in-time digital printer (step 328) with the samebarcode data associated with the tissue block. The tissue sections aretransported from the microtome to the slide sections and are placed onthe glass slide with the printed label (step 330). Optionally, acomparison can be done between a baseline image of the tissue block andan image of the tissue section in step 331. At this point, an error-freeassociation is established between the barcode of the block from whichthe section was cut, and the label on the slide containing that section.Finally, the barcode data on the glass slide can be scanned (step 332)to validate the slide barcode to the block barcode. In some embodiments,the blockface LIMS data, the transfer media location marking, and theslide barcode data are associated (step 334) in order to ensure that thethree pieces of information precisely match, thus ensuring tracking ofthe cut tissue specimen within the automated tissue sectioning device,assuring regulatory quality compliance.

System Implementation

The quality control analyses described above can be achieved using anautomated apparatus for automated transfer of tissue sections from thesample block to transfer medium such as tape and from the transfermedium to slides. In some embodiments, an automated tape transfer systemis provided including a controller, a support for holding a sample blockof tissue embedded within an embedding medium, a cutting deviceconfigured to cut tissue sections from the sample block and a transfermedium for transporting the cut tissue section from the sample block. Aquality control system includes one or more imaging devices configuredto take at least a first image of the sample block and at least a secondimage of the cut tissue section, the first and second images compared toconfirm the cut tissue section corresponds to the pre-sectioned tissueof the sample block, e.g., is within a pre-set criteria.

In some embodiments, an automated method is provided for transferringcut tissue sections from a tissue sample block and providing for qualitycontrol. The method includes the steps of:

-   -   a) advancing a transport medium in an automated system;    -   b) cutting a first tissue section of the sample block;    -   c) transporting the first tissue section away from the sample        block, wherein the cutting exposes a next cutting face of the        sample block;    -   d) transferring the first tissue section to a first slide; and    -   e) comparing the first tissue section to the sample block to        determine if a correspondence is present.

In some embodiments, the automated method includes transporting the cuttissue section to a slide station containing a first slide for transferto the first slide, wherein the image of the first cut tissue section istaken on the first slide

FIG. 23 provides a work flow diagram for section tracking. In step 340,the workflow involves scanning the tissue block and determining if thesample block ID is readable with a scanner (step 342). If not, then adetermination is made if the block ID is readable by a histotechnicianin step 344. If not, the block is flagged in step 346; if yes, the blockID is typed manually on the system in step 348. Once the block ID isreadable (step 350), either by scanning or manual input, the tissue issectioned to create slides as described herein (step 352). If adetermination is made that the tissue on the slides have blade nicks,bubbles, missing parts, and/or other unacceptable features as describedabove (step 354), more sections are cut. If the tissue on the slides isacceptable, then print labels are requested for the slides in step 356.A determination is made about the functionality of the slide labelprinter in step 358. If the slide label printer is not functional, thenthe lab needs to execute a process to get labels in an alternate way(step 360). If the printer is functional, then the labels are printedand affixed to the slides in step 362. Next, the tissue on the slide andthe block face are compared (utilizing the methods/process/systemsdescribed herein) to determine if there is a match, in step 364. If not,a determination is made if any other processed block tissue shapematches the tissue on the slide in step 366. If not, the slide isflagged in step 368; if yes, then a check is made to confirm the slideand sample block barcodes match in step 370. If not, the slide isflagged in step 372; if yes, then the slide is acceptable for processing(step 374).

In reference to FIG. 24, the vision system of the present disclosure canbe a part of an automated microtomy device. In some embodiments, anautomated microtomy device 400 can include a combination of mechanism toreceive a sample block, cut a sample/section from a sample block,transfer a sample cut from the block onto a tape to be transferred to aslide for analysis. The combination of mechanism can include at leastone microtome 404, tape transfer apparatus 406, slide adhesive coater412, a slide printer 414, slide input racks 416, a slide singulator thatpicks a slide from a stack of slides 418, and slide output racks 420.This combination of mechanisms works together to prepare the sample onthe slide and prepare the slide itself.

FIG. 25 is a schematic view of an exemplary embodiment of an automatedtape transfer apparatus (system) 430 that includes a visualizationsystem having an illumination system and an image system. Note otherautomated apparatus could be utilized, and that the apparatus 430 isshown by way of example. FIG. 25 illustrates the path of the tape 432for transporting cut tissue sections after the block is fully faced.FIG. 25 shows a microtome 434 that is used to hold the sample blocks andcut the sections. The microtome 434 holds a sample block comprising atissue sample that is enclosed in a supporting block of embeddingmaterial such as paraffin wax. The microtome 434 includes a blade (notshown) aligned for cutting slices (or sections) from the face of thetissue block.

In addition to the adhesive tape 432 and the microtome 434, theautomated tape transfer apparatus 430 of FIG. 25 includes a motorizedfeed mechanism 436, a tape applicator 438, a slide station 440 and atake-up mechanism 442. An illumination system 444 and imaging system 446for the block face are shown (schematically) in the drawing. The same ora different illumination and imaging system (not shown) can be utilizedfor the tissue section on the tape. An illumination system 448 andimaging system 450 for the slide are also shown (schematically) in thedrawing. The path of the tape 432 starts at the feed mechanism 436 andtravels toward the microtome 434 and an applicator end of the tapeapplicator 438. The tape 432 then travels away from the microtome andtoward the slide station 440 and finally is stored on the take-upmechanism 442. Note details of the apparatus/system 430 are described inU.S. Publication No. 2017/0205317, and U.S. Publication No. 2017/0328818the entire contents of these applications are incorporated herein byreference. The motorized reels advance the adhesive tape so that theportion of the adhesive tape that includes the cut section moves awayfrom the microtome and sample block and a new portion of the adhesivetape is positioned and adhered to the cutting face for the next sectionto be cut by the microtome and transferred to the adhesive tape.

FIG. 26 shows the tape applicator as the cycle begins. The tapeapplicator moves towards the cutting face of the sample block of tissue.This causes the roller member of the tape applicator to press the tape,e.g., the adhesive side of the tape if an adhesive tape is utilized,onto the cutting face to cause the tape to adhere and cover the entirecutting face with tape. The tape applicator is then retracted in theopposite direction causing the roller member to reset to the originalposition of where the roller member is clear of the cutting face. Insome embodiments, the cut tissue section is moved into contact with thetape after sectioning by the microtome.

FIG. 26 shows the slide station 440 of the automated tape transferapparatus 430 in more detail. The slide station 440 can be a UV stationfor transfer of the tissue sections that are on the tape to microscopeslides 460 that are pre-coated with UV-curable adhesive. A roller maythen press the section on the adhesive tape onto the slide. It should beappreciated that although the system of FIG. 25 includes a slide stationfor transfer to slides, the system in some embodiments does not includea slide station and after transfer of the cut sections to the tape andmovement of the tape from the microtome area, the sections can betransferred from the tape to the slides in accordance with othermethods, e.g., manual transfer or stored on the tape.

The slide station 440 has a lower portion 462 with spacers that createthe slide slots, a support section 464, a UV source 466 and a motor 468.The slide slots created by the spacers and the support section 464 holdthe slides 460. The motor 468 is used to translate or move the lowerportion of the slide station 440 to adjust the section location on aslide 460 so that the exact location of where the sample section fromthe tape is deposited on the slide 460 can be controlled. Theillumination and imaging system can be provided in or adjacent the slidestation for illuminating and taking mages of the tissue section on theslide for quality control, e.g., comparison to base images of the tissueprior to transfer to the tape. The imaging system can also be utilizedto assess the condition of the tissue section on the slide for integritychecking of the tissue. FIG. 27 illustrates is an exemplary schematicview showing the tape 432 prior to being applied to a face 470 of asample block 472.

As noted above, the illumination and imaging systems disclosed hereincan be utilized with other automated apparatus, tape other than adhesivetape, and apparatus not having an automated slide station as well as inmanual systems.

The automated systems provide for using an adhesive tape, oralternatively another transfer medium, to support samples from tissueblock cutting. The automated systems and methods also provide forautomated subsequent transfer of the samples from the adhesive tape toslides. The systems and methods further provide for improved qualitycontrol by providing a method and device/system for comparison of i) thetissue on the transfer medium and/or ii) the tissue on the glass slide,to the tissue on the sample block or the slice just cut from the block.This is in the context of a fully automated tissue sectioning device andprovides automated quality control.

The system is described with use of a continuous strip of adhesive tape,it being understood that other transfer medium can be utilized. Theadhesive tape as disclosed herein adheres to the cutting face of thesample block prior to sectioning. Subsequent to the adhesive tapeadhering to the cutting face, the microtome begins a cutting action. Theadhering of the adhesive tape to the cutting face supports the sectionthat is being cut by the microtome. Once the microtome completes thecut, the section that has been cut remains adhered to the adhesive tape.In alternate embodiments, the section can be cut first, followed byadherence to the transfer medium.

Note the tape provides one example of a transport device/system for thetissue section. Other transport systems can also be utilized such arobotic arm, a series of cups with water in them, etc. The tissue in/onthese other transport systems can be evaluated in accordance with thequality control systems described herein. Therefore, the systems, e.g.,illumination systems, imaging systems, etc. described herein are fullyapplicable to the section (slice) on the various transport systems.

Due to the analysis of the tissue at different stages in the apparatus,e.g., block to new slice, slice on transport, slice on slide, multipleinternal levels of quality control are provided.

The slides in the slide station can be stably (firmly) held inaccordance with some embodiments of the quality control system describedherein. In some embodiments, the automated system further includes asupport to hold the sample block stably and a support to hold the slidestably in front of the one or more imaging devices.

It should be understood that the term “adhesive tape” as used hereinrefers to any type of bonding, including molecular bonding, mechanicalbonding, etc., and also can include dry adhesive tapes which providesbonding via van der Waals force (molecular bonding) and whose tape peelforce varies greatly on peel angle which minimizes section damage duringpeeling. The tape can leave no residue, sticks when needed and peels offwhen needed without damaging the tissue. It should also be noted thatthe term “continuous strip of adhesive tape” as used herein means thatthe tape is longer than the amount of adhesive tape used for a singlesection (a single sample of tissue cut from the tissue block). Theadhesive region of the adhesive tape can be large enough to fully coverthe cutting face of the sample block, i.e., to hold a complete sectionwhen it is sliced from the sample block.

An example of an automated apparatus is illustrated in FIG. 26 describedabove, and further described in commonly assigned U.S. Publication No.2017/0205317. Other examples of an automated apparatus, and variationsthereof are disclosed in U.S. Publication No. 2017/0003309 and U.S.Publication No. 2017/0328818. The entire contents of these threepublications are incorporated herein by reference. It should beunderstood that these automated apparatuses provide examples ofautomated apparatus as the illumination/imaging systems and the qualitycontrol systems can be used with other automated apparatus. Also, asdiscussed herein, the illumination/imaging systems and the sectiontracking and quality control systems can be used with manual systems andmethods.

The automated tape transfer apparatus may include a programmable digitalcontroller, a processor or other type of application specific integratedcircuit (ASIC) that is used to control the motion of the automated tapetransfer apparatus 1, communicate with users of the automated tapetransfer apparatus 1 and/or communicate with the microtome 4 to whichthe automated tape transfer apparatus 1 is connected. There are manymotions that can be controlled within the automated tape transferapparatus 1. Examples of these motions include the movement of the feedmechanism 3 and the take-up mechanism 6, movement of the lower portion30 and the translation portion of the slide station 5, movement of thelinear actuator member etc. The controller may also provide informationto users of the functions or conditions of the automated tape transferapparatus 1 such as the number of slides that have been prepared, thenumber of sections that have been transferred, the amount of taperemaining on the roll, etc. The controller is capable of receiving anytypes of input (e.g., mechanical, visual, electrical, etc.) to performits control functions. The controller can also in some embodimentscontrol the quality control systems described herein.

In some embodiments, the automated tape transfer apparatus 1 furtherincludes an optical device to inspect the sample block. For example, themicrotome 4 may store multiple sample blocks for sectioning. The opticaldevice may be used to assess the condition of the cutting face ordetermine the location of the tissue within the embedding medium. In oneexample, a macro image of the cutting face may enable more preciseplacement of the adhesive tape 2 on the cutting face. Analysis of thecutting face may facilitate automatic trimming of the cutting face toexpose the desired tissue for sectioning.

In some embodiments, one or more optical sensors may be used to providefeedback to the controller on the position and quality of the section onthe adhesive tape 2. For example, a brightness sensor in close proximityto a backlit section of the adhesive tape 2 may distinguish between anempty portion of the adhesive tape 2 and a portion that is carrying asection. This may provide an approximate location of the section on theadhesive tape 2 that may be used as an input to the controller forvarious purposes, such as motion control. A CCD imager or similar devicemay be used to image the section to provide feedback on the quality ofthe transfer. These images may be used to check for errors in theprocess, such as incomplete transfer of a section, misalignment of asection on the adhesive tape 2, presence of section trimming waste ontape, etc. In these error cases, additional sections may be taken toreplace defective sections.

A similar optical method of inspecting the section on a slide may alsobe used. A sensor system may provide feedback of the quality of thesection transfer to a slide and alert the controller to errors in theprocess. The same or different optical sensors may be used for both tapeand slide inspection.

The automated tape transfer apparatus may also include in someembodiments an automated system to label slides and sample blocks with abarcode or other moniker for identification. Viable slide labelingmethods include attaching an adhesive printed label, etching a labelinto the material or printing a label onto a dedicated location. Thelabel may link a slide to relevant information such as the originatingtissue block and sectioning date. Sample blocks may be similarlylabeled. To accommodate pre-labeled blocks, an optical reader, such abarcode reader may be used to read the block label to produce therelevant slide labels.

The system can also include an automated quality control system forcomparison of cut tissue to tissue on the sample block to ensure the cuttissue is properly labeled on the slide to match the sample block.

Note the use herein of the term tissue sections or cut sectionscontemplates that initial sections cut from the sample block may notcontain much tissue as they could contain the overlying material, e.g.,paraffin or other embedding medium. However, it is the sections oftissue, i.e., the tape regions containing sufficient tissue sections,that are critical for histopathology and these are among those selectedfor transfer to the slides. A feature to ensure this can be provided inthe manner described herein.

The tape transfer apparatus (system) can include one or more automatedimaging devices such as digital cameras for taking photos during variousstages of the automated tape feed/advancement process. The photos can betaken at the time of the cut section transfer to the tape, at the timeof transfer of the cut section to the slide, and/or at any other timeduring the process. Such photos can provide visual/quality control asdescribed herein.

Photos can also be taken of the sample blocks (block face). For example,a mismatch between the block face image and section on tape image is acue for an error during sectioning. A macro image can be useful in athumbnail in a database listing section images. This can be useful forroughly figuring out when to start transferring sections to the tapewhen cutting. These are various ways to image the tissue within thesystem other than a digital camera. For example, MicroCT can be used toconstruct a 3D model of the tissue within the paraffin. If the systemhas a 3D model of the tissue in the block as input, it could use theinformation to determine when to stop trimming and sectioning.

In another aspect of visual/quality control, as the tape advancesthrough the apparatus and sections are cut from the sample block by themicrotome and adhered to the adhesive of the tape, a photo (or otherimaging technique) is taken of each tape region containing a tissuesample (cut section) transferred to the adhesive tape thereby enablingreal time analysis to make sure the section has been properly, i.e.,completely, transferred to the tape. Utilizing the same camera orimaging device, or alternatively, utilizing another camera or imagingdevice, as the tape with the adhered sections cut from the sample blockadvances to the slide station and the section is transferred to a slide,a photo (or other imaging technique) is taken of each slide containingthe sample to enable real time analysis to make sure the section hasbeen properly, i.e., completely, transferred to the slide. In thismanner, the process can be monitored to ensure adequate sections of thesample block are cut and transferred to slides for pathology beforecessation of the tape feed. In certain embodiments, if inadequatesections have been transferred, the system can be reversed and the tapeunwound in the direction opposite the initial direction of advancementto collect and transfer more sections (samples) from the sample block.Also note that multiple photos of each tape region and each slidecontaining the cut section can be taken for evaluation. The illuminationand imaging systems described herein enhance this analysis.

Other information from the photos can also be stored to identify thesample block, sections, etc. in conjunction with the marking andtracking of the blocks and sections.

In the implementation of the visual control system, a photo is taken ofeach tape region containing a section, e.g., tissue section, cut fromthe sample block by the microtome. The photo is then analyzed todetermine whether the cut section is properly transferred to the tape.In a further analysis, the photo is evaluated to determine the end ofthe sample block trimming (described below). A photo is also taken ofthe slide once the tissue section has been transferred to the slidewithin the slide station (downstream of the microtome). This photo ofthe slide is analyzed to determine if the tissue section was properlytransferred to the slide. The photos can also be analyzed to determineif sufficient tissue sections are contained on slides. The photos canalso be utilized for matching to the sample block. If the section is notsufficient, e.g., it does not contain a sufficient tissue sample as aresult for example of containing mostly paraffin, the section is notused for evaluation. The illumination and imaging systems describedherein enhance these analyses. The illumination and imaging systemsdescribed herein enhance these analyses.

The photos can be stored in a database for future selection if furtheruse and analysis is desired.

FIGS. 28A, 28B, 28C, 29, and 34 illustrate an example embodiment of anautomated system for the implementation of the above-described methods.It should, however, be noted that the methods and systems describedabove can be implemented in a manual microtomy process or otherautomated microtomy processes.

Referring to FIGS. 28A and 28B, in some embodiments, an automated system500 is provided to enable automated tissue sample processing from blockto slide. The system 500 can be designed to include a first section forcutting samples from the tissue blocks. In some embodiments, the firstsection, for example as shown in FIG. 28B, can include a block handler,at least one microtome 504, a transfer medium 506 (e.g., a tape), ahydration chamber 508, and a block tray 510. The block handler, the atleast one microtome 504, the transfer medium 506 (e.g., a tape), thehydration chamber 508, and the block tray can be designed to operatetogether to organize, face, hydrate, section biological samples fromtissue blocks and transfer the tissue sections to slides using anycombination of systems and methods.

In some embodiments, the system 500 can include a transfer medium 506(e.g., a tape) to receive the sample slices taken from the tissue block,for example, by a sectioning microtome 504. The transfer medium 506 caninclude any combination of materials or surfaces that are able toreceive a sectioned sample from a microtome 504 and transport thesectioned sample to another location. In some embodiments, the transfermedium 506 can include at least one adhesive surface capable ofremoving, receiving, and/or transporting a sectioned sample from amicrotome 504 after it has been cut from the tissue block. For example,the transfer medium 506 can include any combination of tapes, such asfor example, a tape roll, windowed tape, etc. The transfer medium 506can include or otherwise be a part of a larger mechanism fortransferring a sectioned sample. For example, the transfer medium 106can be an adhesive tape wrapped over a combination of pulleys, wheels,spools, conveyers, etc. designed to enable the transfer medium 506 movea sectioned sample thereon from one location to another. Any othercombination of transfer mediums can be used without departing from thepresent disclosure. For example, the transfer medium 506 can be a beltwith ridges, dips, etc. designed to grasp and/or hold the sectionedsamples.

In some embodiments, the transfer medium 506 can transfer sectionedsamples from its surface to a shifting assembly 522 for transferring thesample onto a slide. The shifting assembly 522 can be designed to removethe samples adhered to the transfer medium 506 and place the samples onone or more slides. In some embodiments, transferring by the shiftingassembly 522 can include separating the actual tissue sample material toisolate the sample from the non-sample material. The shifting assembly522 can use any combination of systems or methods to separate anentirety or a portion of the biological sample for the surrounding theparaffin material such that only the biological sample material istransferred to the slides. For example, the shifting assembly 522 cancore out a portion of the biological sample to be removed from thetransfer medium 506. In some embodiments, the non-sample material (e.g.,paraffin material) can remain on the transfer medium 506 to be discardedwith the used transfer medium 106.

Continuing with FIGS. 28A-28C, the system 500 can also include a secondsection, for example as shown in FIG. 28C, having a combination ofmechanisms to prepare and provide a slide to receive a biological samplecut from the block (e.g., in the first section) from the transfer medium506 (e.g., a tape) and processing the slide for analysis. In someembodiments, the combination of mechanisms for processing the slide inthe second section can include a slide adhesive coater 512, a slideprinter 514, slide input racks 516, a slide singulator 518, and slideoutput racks 520. This combination of mechanisms can work together toprepare the slide to receive a sample, secure the sample on the slide,and deliver/organize the slide with the sample to a rack for later use.In some embodiments, initial blank slides can be provided within astorage rack of a slide assembly for pre-processing. For example, theslide assembly can include one or more slide input racks 516 for storinga plurality of blank slides. The slide assembly can store and organizeslide in a large capacity, for example, 200 slides.

In some embodiments, the slide singulator 518 can be designed to grab aslide from a stack of slides within the input racks 116. The slidesingulator 518 can includes any combination of mechanisms capable ofpicking up and transporting the slides. For example, the slidesingulator 518 can be an actuating mechanical arm, a gantry, etc. Beforebeing processed, the slide singulator 518 can provide slides for aquality control step. During the quality control step, an analysis canbe performed on the slide to ensure the slide is suitable to receive asample. For example, the quality control can include the slidesingulator 518 transporting the slide in view of a camera to provideimage data for image processing to identify any potential issues withthe slide, check an orientation of a slide, a condition of a slide, etc.If the slide fails the quality control inspection it can be discarded,if it passes, it can be transported within the system 500 to be preppedto receive a sample. In some embodiments, the slide can be transportedto the slide printer 514 to receive an identification informationprinted thereon. For example, information about a sample type, sampleorigin, sample date, etc. can be printed on the slide. Theidentification information can include any combination of machinereadable and human readable code or text such that the slides and thecontent thereof can be properly identified and tracked. For example, theslide printer 514 can print a machine-readable barcode on the slide toidentify the slide number, batch, contents, etc.

In some embodiments, the slide can be transported to the slide adhesivecoater 512 to be coated by an adhesive material. For example, the slideadhesive coater 512 can spray an ultraviolet (UV) activated adhesive onthe slide, apply an UV activated adhesive tape, or any combination ofadhesive systems or methods. In some embodiments, the adhesive can beapplied in multiple layers. The numerous layers can be applied such thatthe slide receives a uniform coating of the adhesive to ensure clearviewing through the slide layer. In some embodiments, the slide can beinserted into the slide input racks 516 already preprocessed orpartially pre-processed.

Once the slide has been processed by the slide printer 514 and the slideadhesive coater 512, the slide can be transported to the transfer medium106 to receive a sample from the transfer medium 506. For example, theslide can be transported to the shifting assembly 522 to receivesectioned tissue block samples from transfer medium 506 (e.g., a tapemechanism). In some embodiments, prior to transferring the sample to aslide, the shifting assembly 522 can include one or more cameras toperform image processing to determine whether samples of the transfermedium 506 are suitable for adhesion to a slide. For example, the imageprocessing can inspect the sample to determine whether it is suitablefor placement on a slide. If it is not suitable the sample can bediscarded and the transfer medium 506 can be advanced to the nextsample. When a sample is suitable for placement on a slide, it can beapplied to the slide. In some embodiments, the image processing caninspect the sample after it has been adhered to the slide to determinewhether or not the placement of the sample is of sufficient quality. Forexample, the image processing can inspect the slide to determine whetherthe sample is cleanly adhered to slide, no bubbling, tearing, paraffinremanence, etc. If a slide is not suitable, the slide can be discardedinstead of being placed in the slide output racks 520.

In some embodiments, the completed slides can be transported, by thesingulator 518, to be stored in the slide output racks 520. The slidescan be stored in the slide output racks 520 in a predetermined orderand/or organizational method such that the next steps in which theslides will be used can easily locate and remove the slides.

As noted above, in some embodiments, the system 500 can include aquality control imaging system as disclosed, for example, in co-pendingU.S. Application No. 62/980,203, filed on Feb. 22, 2020, which isincorporated herein by reference in its entirety.

Referring to FIG. 29, in some embodiments, system 500 can be used totransfer samples from tissue blocks to slide following the stagesprovided in the automated process flow 600. FIG. 29 shows the processflow of a block to slide steps used in the system 500 provided in FIGS.28A-28C. At step 601, the sample tissue blocks can be loaded in thesystem 500. For example, one or more tissue blocks, with tissue samplesembedded within a paraffin block, can be loaded into trays 510 andplaced within the system 500. At step 602, one of the sample tissueblocks can be moved from the tray 510 to a microtome 504 for facing. Forexample, a tissue block can be transported by a handler and placedwithin a chuck of a facing microtome 504 to be faced. At step 603, thefaced tissue block can be moved to the hydration chamber 508 to behydrated and cooled. For example, a tissue block can be transported by ahandler and placed within the hydration chamber 508 for a predeterminedperiod of time. After sufficient hydration has been provided, at step604, the tissue block can be moved to a microtome 504 for sectioning.For example, a tissue block can be transported by a handler and placedwithin a chuck of a sectioning microtome 504 to be polished andsectioned. The block can be provided to the same microtome 104 thatperformed the facing or a different microtome 504. Thereafter, eachsectioned sample can be transferred to the transfer medium 506. At step605 the sectioned samples on the transfer medium 506 can be transferredto a slide.

Simultaneous to or subsequent to steps 601-605, steps 606-608 can beperformed to prepare one or more slides for combining with the sectionedsamples from the tissue block. At step 606, a microscope slide can beselected and obtained from a stack of new slides. For example, the slidesingulator 518 can select and pull a slide from a stack of slides storedwithin a rack 516 of blank slides. At step 607, identifying informationcan be printed on the selected slide. For example, the slide can beplaced within the slide printer 514 to have a machine-readable barcodeprinted thereon. At step 608, an adhesive material can be coated on theselected slide. For example, the slide can be placed within the slideadhesive coater 512 to have a UV activated adhesive sprayed thereon. Atstep 609, the tissue sample can be transferred from the transfer medium506 to the UV adhesive coated slide. Additionally, during step 609, theslide can be imaged for onboard diagnostics, quality control, and sampletracking. For example, one or more cameras can be used to capture imagedata to be processed by an image processor for predetermined qualitythresholds. Once the slide has passed the quality control, at step 610the completed tissue slide can be moved to the output rack 520 to bestored for future analysis.

Algorithms

The flow chart of FIG. 30 illustrates the steps of the motor controlledautomated systems for transferring a tissue section to tape cut by amicrotome and further transferring the tissue section to a slide. Forexample, the automated system can include a movable tape or othersupporting/carrying medium, and the sections of tissue are automaticallytransferred by the apparatus to a tape. Once the system, imaging systemdetermines that the block has been fully faced as described above sothat tissue sections can be transferred to the tape for later analysis,the feed mechanism is automatically activated (or alternatively theapparatus can be designed that once the block has been fully faced, theuser would activate the feed mechanism). Activation of the feedmechanism advances the tape which is moved toward the cutting face ofthe sample block as described above. Next, the roller, e.g., rollermember, presses the tape, e.g. an adhesive side of the tape if anadhesive tape is utilized, onto the cutting face. The roller is thenpushed down so the tape covers the entire cutting face. The linearactuator is retracted to its original position to reset the roller forsubsequent application of tape to the block for transfer of another cutsection. The microtome then cuts the section covered by the tape (e.g.,along a plane parallel or substantially parallel to the cutting face).The cut section carried by the tape is advanced to the slide station toalign with the slide. After the cut section of tissue is transferred bythe automated apparatus to the tape, the tissue section is subsequentlytransported by the automated apparatus to a glass slide at the slidestation and automatically transferred to the glass slide. The slideroller presses the section on the tape onto the slide, and the sectioncan be laminated onto the slide. The slide roller is retracted to itsoriginal position and the tape is advanced away from the slide, leavingthe section on the slide. These steps of FIG. 30 repeat until a desirednumber of sample sections have been transferred to the tape, cut by themicrotome and transferred to slides.

As shown in FIG. 30, the feed mechanism is activated to advance the tapein step 700. Next, the linear actuator moves toward the cutting face ofthe sample block in step 702. The roller member presses the adhesiveside of the tape onto the cutting face in step 704. The roller memberthen pushes down to adhere the adhesive tape to cover the entire cuttingface in step 706. The linear actuator retracts to reset the rollermember for subsequent application of the adhesive in step 708. Themicrotome cuts the section covered by adhesive tape in step 710, and thecut section advances to the slide station to align with the slide instep 712. The slide roller presses the section onto the slide in step714, and the section is laminated onto the slide in step 716.Optionally, a comparison can be made between a baseline image of thetissue block and an image of the tissue section on the slide in step717, to ensure a match between the tissue block and the tissue section.The slide roller retracts to its original position in step 718. Finally,the tape advances away from the slide and is stored on the take upmechanism in step 720.

In some embodiments, a quality control system can be provided for anequipment or component check of the automated system. More specifically,a software algorithm can be utilized to determine if the tissue transfersystem is working according to the manufacturing specifications based onthe variation of the tissue images and the projected pattern. This canbe based for example on images of the tissue on the tape. In analternate variation, landmark features of the fixed machine componentscan be used as a reference instead of the projected pattern to determinetissue orientation variations. Such feature may help with devicepredictive maintenance. The tissue is transferred to tape at the samenominal location if everything is working according to thespecifications. If the tissue transfer is at a sufficiently differentlocation, it can alert the user that machine components are misalignedor not working properly. For example, if a different location isdetected, this could mean the rollers need to be aligned, the tapetension needs to be the same between transfer, e.g., the tension sensoris dislodged or the sensor is tripping at a different point, the tape toblock applicator tension spring still does not have the specified springconstant, the tape used for transfer does not have the same elasticityconstant and specified, etc. It is understood that a single metriccannot point to any individual reason, but it can alert the technicianthat there is an issue that needs to be fixed in the tissue transferline. It is also contemplated, however, that the system can provide analgorithm that can detect with more specificity the source ofnon-alignment and therefore the machine component(s) that needsadjustment can be identified.

The tissue imaging systems are shown in use with the tape transferapparatus (system) of FIG. 25 with the flow chart of FIG. 31illustrating the steps of the automated tape transfer system. Note asingle imaging device, e.g., a digital camera, can be utilized to takephotos adjacent the transfer of the cut section to the adhesive of thetape. The same cameras can be repositioned during the automatedoperation to adjacent the slide station to take photos of the slidesafter transfer of the section to the slide. Alternatively, a differentimaging device can be provided within or adjacent the slide station totake photos after transfer of the section to the slide. As noted above,the apparatus of FIG. 25 can take photos of the cut sections aftertransfer of the cut sections to the tape and after transfer of the cutsections from the tape to the slides, or, alternatively, take photosonly after transfer to the tape or only after transfer to the slide.Such photos can be taken at the time of transfer, right after transferor downstream of the transfer (after the tape has advanced past the tapeapplicators or advanced to the slide station). The photos of the tapeand/or cut sections of the tape can also be taken at other times duringthe tape feed cycle if desired.

First, the feed mechanism is activated to advance the tape in step 730.The linear actuator moves toward the cutting face of the sample block instep 732. The roller member presses the adhesive side of the tape ontothe cutting face in step 734, and the roller member pushes down toadhere the adhesive tape to cover the entire cutting face in step 736.The linear actuator retracts to reset the roller member for subsequentapplication of the adhesive in step 738. The microtome cuts the sectioncovered by the adhesive tape in step 740. A photo is taken of the cutsection covered by the tape in step 742, and the cut section advances tothe slide section to align with the slide in step 744. Next, adetermination is made about tissue sufficiency in step 746. If thetissue section is not sufficient for transfer onto the slide, thesection is not transferred to the slide and remains on the tape in step748. The tape then advances away from the slide and is stored on thetake up mechanism in step 758. If the tissue section is sufficient fortransfer onto the slide, the slide roller presses the section onto theslide in step 750, the section is laminated onto the slide in step 752,and a photo is taken of the section on the slide in step 754. Finally,the slide roller retracts to its original position in step 756 and thetape advances away from the slide and is stored on the take up mechanismin step 758.

With reference to the flow chart of FIG. 31, after the tape, e.g., thetape cartridge, is loaded onto the feed mechanism, the feed mechanism436 is activated to advance the tape, i.e., a continuous length ofadhesive tape. The linear actuator member 438 is moved toward thecutting face of the sample block. Next, the roller member presses theadhesive side of the tape onto the cutting face. The roller member isthen pushed down to adhere the adhesive tape to cover the entire cuttingface. The linear actuator 438 is retracted to its original position toreset the roller for subsequent application of adhesive tape to anothersample. The microtome then cuts the section covered by the adhesive tape(along a plane parallel or substantially parallel to the cutting face).A photo is taken of the cut section by the digital camera, either at thetime of transfer or right after the transfer. The photo is analyzed toconfirm proper transfer to the tape. The cut section is advanceddownstream to the slide station 440, to align with the slide. At thistime the photo is analyzed to determine if a sufficient section oftissue has been cut from the sample block for transfer to the slide. Ifthe section is not sufficient, e.g., it does not contain a sufficienttissue sample as a result for example of containing mostly paraffin, thesection is not transferred to the slide and remains on the tape. If thetape region contains a sufficient tissue section, then it is ready fortransfer to the slide and the slide roller presses the section onto theslide and the section is then laminated onto the slide by the variousmethods described above. A photo is taken of the cut section and slideat the time of transfer to the slide or right after the transfer. Notethe photo can be taken before or after lamination onto the slide. Notethe slides from the slide machine have a bar code or otheridentification system corresponding to the bar code or other identifieron the sample block. The slide roller is retracted to its originalposition and the tape is advanced away from the slide and stored on thetake up reel of the tape cartridge mounted on the take up mechanism.These steps of FIG. 31 repeat until a desired number of sections fromthe sample block have been cut by the microtome, transferred to thetape, and transferred to slides. Photos are taken of each of thesesections when transferred to the tape and when transferred to the slidefor analysis during the tape feed operation (quality control).

The flow chart of FIG. 32 is for a system similar to that of FIG. 31except the sample tape is removed from the carrier strip as in thesystem of U.S. Publication No. 2017/0003309. The analysis of the photosin accordance with the flow charts of FIGS. 31 and 32 are enhanced byuse of the illumination and visioning systems described herein.

As shown in FIG. 32, the feed mechanism is activated to advance thecarrier strip carrying patches of the sample tape with the adhesive instep 760. Next, the sample tape aligns with the sample surface in step762. The roller moves to press the sample tape onto the sample surfaceand the carrier strip guide is now in the Apply position in step 764.The roller retracts to its initial position in step 766. The carrierstrip guide moves to the Remove position to move the carrier strip outof the path and the carrier strip separates from the sample tape in step768. The microtome cuts a section of the sample in step 770 and a photois taken of the cut section on the tape in step 772. Next, the sampletape with the attached section advances to the slide station to alignwith the slide in step 774. Next, a determination is made about tissuesufficiency in step 776. If the tissue section is not sufficient fortransfer onto the slide, the section is not transferred to the slide andremains on the sample tape in step 778. If the tissue section issufficient for transfer onto the slide, the slide roller presses thesection onto the slide in step 780, the sample tape is removed in step782, and the section is laminated onto the slide in step 784. Finally, aphoto is taken of the section on the slide in step 786.

As discussed above, the automated system has an image based qualitycontrol system to compare the tissue on the block face of the sampleblock with the tissue sections transferred to the glass slides. One ormore imaging devices acquires digital image(s)s of the block face andone or more imaging device(s) acquires digital images of the tissuesection on the slide onto which the tissue section is transferred andretained, and the images of the sample block and slide are compared topositively match the items. That is, the images from the sample blockand the images from the slides containing the tissue sections arecompared to determine the presence or absence of a match. This providesa backup system to the barcodes which are provided on the cassettecarrying the tissue block and on the slides. In this manner, if thematching of the barcodes is confirmed, a double check is performed bythe automated apparatus by the image comparison. Thus, quality controlis not reliant on human assessment.

To achieve such quality control, in some embodiments, three features areprovided: 1) a series of image capture devices placed to capture thedesired tissue images; 2) contrast is created to improve differentiationon the digital images between the tissue and paraffin (or otherembedding medium) to facilitate comparison/analysis; and 3) the tissueblock and the slides are stably (firmly) held to minimize, or in someembodiments fully prevent, movement of the block and slide whichenhances imaging. Each of these features is discussed below.

The automated system can include a computer system to collect the imagesfrom the block facing camera and the slide facing camera with thesoftware comparing the images from the block face and the slide. Thesoftware algorithm determines the outline of the tissue from each imageand compares the two images. The images can be stored for latercomparison if desired.

The computer system can have a decision algorithm to determine if theimages from the block face and the slide are matching or not. Thedecision algorithm has the knowledge of identification of the sampleblock and of the glass slide from which the images are captured. Thedecision algorithm verifies that the samples also have matchingidentifications. The matching identifications can involve matchingtissue contours as described herein. The matching identification canalso involve matching bar codes on the sample block and on the slides,as shown for example in FIGS. 17A and 17B, described herein.

Various types of imaging devices can be provided. Note the terms“imaging device” and “image capturing device” are used hereininterchangeably and for convenience are discussed and shown in terms ofa digital camera, however, it should be understood that various devicesand methods for capturing the images are also contemplated including forexample, X-ray, infrared, tomography, microCT imaging, OCT camera, etc.A single image device can be provided, but alternatively multipleimaging devices are provided adjacent the sample block and adjacent theslide receiving the tissue section to enhance the image. Note the imagecapture devices, e.g., digital imaging devices such as digital cameras,can have light filters on the incoming light as the image capture deviceacquires images of the block to enhance clarity.

The imaging system could include a box that includes the object to beimaged and the imaging hardware to prevent stray light to be captured bythe camera.

In some embodiments, another aspect to having a clear and a highcontrast image is to minimize the vibrations between the image capturedevice and the object. The automated system can include a mechanism thatholds the sample block of tissue to be sectioned stably in front of aplurality of cameras. This enhances the images when using camerassensitive to vibrations by reducing vibrations of the sample block sincesuch vibrations blur the image and decrease the performance of the imagepost-processing tools by making tissue comparisons more difficult. Thesample block is held in place with servo motors that monitor theposition of the sample in real time. In one embodiment, the sample blockis held in place with high inertia mounting brackets attached to thesame reference frame as the image capture device. Other mechanisms forholding the sample block stably are also contemplated.

For likewise providing a clear and high contrast image of the “second”images, i.e., the cut tissue section on the slides, the vibrationsbetween the image capture device and the slide is minimized. Thus,similar to holding the sample block stable as discussed above, theautomated system can include a mechanism that holds the slide containingthe cut tissue sample stably (firmly) in front of a plurality ofcameras. This stable holding enhances the images by reducing vibrationsof the slide since such vibrations blur the image and decrease theperformance of the image post-processing tools by making tissuecomparisons more difficult. The slides can be held firmly in placewithin the slide station by the slide holder during imaging.Alternatively, a mechanism, such as a mounting bracket, can beincorporated as part of the slide station.

The system and method of the automated system of one embodiment will nowbe described in conjunction with the flow chart of FIG. 33. The methoddescribed is an automated biological tissue sectioning device whichprocesses paraffin embedded biological tissue and produces thin sectionson a glass substrate. The thin sections are suitable for analysis undera microscope after further processing. In the method, the tissue shapeon the block face is compared to the tissue outline on the glass slide.This occurs in the context of a fully automated tissue sectioning deviceand provides automated quality control capability. Thus, the systemprovides an input and output product comparison for an automatedbiological tissue sectioning device, thereby providing quality controlof the biological tissue being cut and placed on a glass slide.

As shown in FIG. 33, a photo is taken of the sample block in step 790and the feed mechanism is activated to advance the tape in step 792. Thelinear actuator moves toward the cutting face of the sample block instep 794. Next, the roller member presses the adhesive side of the tapeonto the cutting face in step 796. The roller member then pushes down toadhere the adhesive tape to cover the entire cutting face in step 798.The linear actuator retracts to reset the roller member for subsequentapplication of the adhesive in step 800. The microtome cuts the sectioncovered by the adhesive tape in step 802, the slide roller presses thesection onto the slide in step 804, and the section is laminated ontothe slide in step 806. An image is taken of the section on the slide instep 808. The slide roller retracts to its original position in step 810and the tape advances away from the slide for storage on the take upmechanism in step 812. The section image is compared to the sample blockimage to confirm a match in step 814. Optionally, a comparison can bemade between a baseline image of the tissue block and the section imagein step 815, to ensure a match between the tissue block and the tissuesection. The bar code on the slide is compared to the bar code on thesample block to confirm a match in step 816.

More specifically, with reference to the flow chart of FIG. 31, thesteps are similar to the system described in conjunction with the flowchart of FIG. 31 except for the addition of the quality control. Thesystem of FIG. 31 also does not have the quality control step ofdetermining whether the tissue should be transferred to the slide,however, the quality control system can be used with a system performingsuch steps. In the initial step of FIG. 31, one or more photos are takenof the sample block by a digital camera. The initial photos can be takenbefore activation or after activation of the feed mechanism. The imagesare stored for comparison to images taken later in the process, i.e.,after transfer to slides (or transfer to tape). Next, the methodincludes: moving the tape toward the cutting face of the sample block,pressing the adhesive tape onto the cutting face to adhere the cuttingface to the surface (for example, the entire surface of the cuttingface), cutting the tissue section via the microtome, advancing the cuttissue section on the tape to the slide station, pressing the tapesection onto the slide, and laminating the section on the slide. Inaccordance with the quality control system, an image is taken of thetissue section on the slide (after before or after lamination) by adigital camera. The sectioned image is compared to the sample blockimage to confirm a match. The bar code on the slide is also compared tothe bar code on the sample block to confirm a match. This sectioncomparison between the sample block and the tissue section on the slideis shown in FIG. 17A wherein the image taken of the top of the sampleblock containing tissue embedded in paraffin is compared to the tissuesection on the slide. The bar code on the individual slide is comparedto the bar code on the back of the sample block as shown in FIG. 17Bwhich illustrates the slide and paraffin block identificationcomparison. Note the bar code comparison can be performed before orafter the tissue image comparison.

Note the steps in the flow chart provide one embodiment for use of thequality control system, it being understood that the steps need not beperformed in the exact order of the blocks of FIG. 33.

It should be appreciated that the quality control system is described inconjunction with the method of FIG. 33 can include a quality controlsystem taking photos of the tape when the tissue section has beentransferred thereto. The quality control system for identificationmatching for positive verification can also be used without this tapesection transfer photo check. The quality control system ofidentification matching can be used with any of the apparatus/systemsand methods described herein.

It should be appreciated that a single camera or multiple cameras (orother imaging devices) can be used for the sample block, cut sectionand/or slide images and or tape images. Additionally, it is alsocontemplated that a single camera (or other imaging device), eitherstationary or movable, can be utilized for taking images of the sampleblock, tape and/or slides.

Note that multiple images of the sample block can be taken to provide a1-1 comparison of the images of each slide. For example, before eachsection is cut an image can be taken of the block face for comparison toa slide containing that specific cut section. Alternatively, only asingle base image or a few base images of the sample block can be takenfor comparison to the images of each slide.

In accordance with another aspect of the quality control system, imagesof the slides are processed to check for bubbles or tears, i.e., lookingfor artifacts to confirm proper transfer to the cut tissue section tothe slide. If there are bubbles, then the slide can be tossed. Note in asecond level of such artifact quality control system, if an artifact isdetected, the system would next determine if the artifact is on thetissue or paraffin. If on the tissue, the slide can be discarded; if onthe paraffin the slide could still be used since it would not affecttissue analysis. This artifact quality control system can in someembodiments be utilized in addition to the sample block/slide imagecomparison quality control system described herein.

As noted above, in the various embodiments disclosed herein, it iscontemplated that in certain applications multiple sections can betransferred to a single slide. It is also contemplated in someembodiments that not all of the sections (or slides) are stained. Forgenetic analysis, tumor specific sections of tissue are typically doneon blank or unstained sections to preserve the DNA since the stain canruin the DNA. However, the contrast between the regular tissue and tumoris very poor as the unstained section is mainly transparent under themicroscope. In the systems disclosed herein, the slide station caninclude in some embodiments alternating stained and unstained slides.That is, by placing an unstained section (slide) next to a stainedsection (slide), and detecting the positioning of the sections on thetape and thus the slides due to the tracking methods disclosed herein,the unstained slides can be genetically analyzed. Thus, the stainedslide which is nearly identical to the unstained slide will provideregions/coordinates to pick material from the unstained slides. This isachievable since typically a cut section is 5 microns thick which isabout ½ the size of the cell.

As described herein, photos are taken at various stages of the tape feedcycle for real time analysis. Such photos can be utilized in addition toor as an alternative to the optical sensors discussed above forproviding feedback of the quality of the section transferred to the tapeand/or the quality of the section transferred to the slide.

Any images gathered, and bar-code associations, can in some embodimentsbe synchronized with the laboratory information management system(LIMS). In acquiring images/spectra or making decisions, theseimages/spectra/decisions can make their way into LIMS. Thus, the qualitycontrol systems disclosed herein can facilitate such integration.

The automated system can include a computer system to collect andanalyze the imaging date collected by the imaging system 2. The imagescan be stored for later analysis or comparison if desired. The computersystem can have a decision algorithm to determine (in a binary analysis)if the images from the block face, tape or slide shown tissueabnormalities.

Any suitable computing systems can be used to implement the computingdevices and methods/functionality described herein and be converted to aspecific system for performing the operations and features describedherein through modification of hardware, software, and firmware, in amanner significantly more than mere execution of software on a genericcomputing device, as would be appreciated by those of skill in the art.One illustrative example of such a computing device 900 is depicted inFIG. 34. The computing device 900 is merely an illustrative example of asuitable computing environment and in no way limits the scope of thepresent invention. A “computing device,” as represented by FIG. 34, caninclude a “workstation,” a “server,” a “laptop,” a “desktop,” a“hand-held device,” a “mobile device,” a “tablet computer,” or othercomputing devices, as would be understood by those of skill in the art.Given that the computing device 900 is depicted for illustrativepurposes, embodiments of the present invention may utilize any number ofcomputing devices 900 in any number of different ways to implement asingle embodiment of the present invention. Accordingly, embodiments ofthe present invention are not limited to a single computing device 900,as would be appreciated by one with skill in the art, nor are theylimited to a single type of implementation or configuration of theexample computing device 900.

The computing device 900 can include a bus 910 that can be coupled toone or more of the following illustrative components, directly orindirectly: a memory 912, one or more processors 914, one or morepresentation components 916, input/output ports 918, input/outputcomponents 920, and a power supply 924. One of skill in the art willappreciate that the bus 910 can include one or more busses, such as anaddress bus, a data bus, or any combination thereof. One of skill in theart additionally will appreciate that, depending on the intendedapplications and uses of a particular embodiment, multiple of thesecomponents can be implemented by a single device. Similarly, in someinstances, a single component can be implemented by multiple devices. Assuch, FIG. 34 is merely illustrative of an exemplary computing devicethat can be used to implement one or more embodiments of the presentinvention, and in no way limits the invention.

The computing device 900 can include or interact with a variety ofcomputer-readable media. For example, computer-readable media caninclude Random Access Memory (RAM); Read Only Memory (ROM);Electronically Erasable Programmable Read Only Memory (EEPROM); flashmemory or other memory technologies; CDROM, digital versatile disks(DVD) or other optical or holographic media; magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devicesthat can be used to encode information and can be accessed by thecomputing device 900.

The memory 912 can include computer-storage media in the form ofvolatile and/or nonvolatile memory. The memory 912 may be removable,non-removable, or any combination thereof. Exemplary hardware devicesare devices such as hard drives, solid-state memory, optical-discdrives, and the like. The computing device 900 can include one or moreprocessors that read data from components such as the memory 912, thevarious I/O components 920, etc. Presentation component(s) 916 presentdata indications to a user or other device. Exemplary presentationcomponents include a display device, speaker, printing component,vibrating component, etc.

The I/O ports 918 can enable the computing device 800 to be logicallycoupled to other devices, such as I/O components 920. Some of the I/Ocomponents 920 can be built into the computing device 900. Examples ofsuch I/O components 920 include a microphone, joystick, recordingdevice, game pad, satellite dish, scanner, printer, wireless device,networking device, and the like.

While the above description contains many specifics, those specificsshould not be construed as limitations on the scope of the disclosure,but merely as exemplifications of preferred embodiments thereof. Thoseskilled in the art will envision many other possible variations that arewithin the scope and spirit of the disclosures.

What is claimed is:
 1. A method for quality control in histology systemcomprising: receiving a tissue block comprising a tissue sample embeddedin an embedding material; imaging the tissue block, prior to removingone or more tissue sections, to generate a baseline imaging data of thetissue sample; imaging the tissue block to create a first imaging dataof the tissue sample in a tissue section of the one or more tissuesections on the tissue block; removing the tissue section from thetissue block, the tissue section comprising a part of the tissue sample;imaging the tissue section to create a second imaging data of the tissuesample in the tissue section; comparing the first imaging data to thesecond imaging data to confirm correspondence in the tissue sample inthe first imaging data and the second imaging data based on one or morequality control parameter; and comparing the first imaging data, thesecond imaging data or both to the baseline imaging data.
 2. The methodof claim 1, wherein the tissue section is non-confirming if there is nocorrespondence in one or more quality control parameters in the tissuesample in the first imaging data and the second imaging data.
 3. Themethod of claim 2, wherein the one or more quality control parametersinclude one or more of shape of the tissue sample, size of the tissuesample, or one or more mechanical damages.
 4. The method of claim 3,wherein the tissue section is non-conforming if there is nocorrespondence in the shape or the size of the tissue sample in thefirst imaging data and the second imaging data.
 5. The method of claim3, wherein the one or more mechanical damages are selected from thegroup consisting of tearing, shredding, blade marks, wrinkling,cracking, bubbles, insufficient tissue sample, incomplete tissue sample.6. The method of claim 5, further comprising identifying asnon-confirming a tissue section if one or more mechanical damages arepresent in the tissue sample in the second imaging data but not in thefirst imaging data.
 7. The method of claim 1, further comprisingtransferring, using a transfer medium, the tissue section to a slide,and the second imaging data comprises an imaging data of the tissuesection on the transfer medium or an imaging data of the tissue sectionon the slide.
 8. The method of claim 7 further comprising comparing atleast two of the first imaging data, the imaging data of the tissuesection on the transfer medium or the imaging data of the tissue sectionon the slide.
 9. The method of claim 1, further comprising illuminatingthe tissue section to enhance a contrast between the tissue sample andthe embedding material in the tissue sample.
 10. A method for qualitycontrol in histology system comprising: receiving a tissue blockcomprising a tissue sample embedded in an embedding material; imagingthe tissue block to create a first imaging data of the tissue sample ina tissue section on the tissue block; removing the tissue section fromthe tissue block, the tissue section comprising a part of the tissuesample; imaging the tissue section to create a second imaging data ofthe tissue sample in the tissue section; and comparing the first imagingdata to the second imaging data to confirm correspondence in the tissuesample in the first imaging data and the second imaging data based onone or more quality control parameters, wherein one or both of theimaging steps comprise illuminating the tissue sample with UV light andimaging the tissue sample with a visible range camera to create thefirst imaging data or the second imaging data.
 11. The method of claim10, wherein the tissue section is non-confirming if there is nocorrespondence in one or more quality control parameters in the tissuesample in the first imaging data and the second imaging data.
 12. Themethod of claim 11, wherein the one or more quality control parametersinclude one or more of shape of the tissue sample, size of the tissuesample, or one or more mechanical damages.
 13. The method of claim 12,wherein the tissue section is non-conforming if there is nocorrespondence in the shape or the size of the tissue sample in thefirst imaging data and the second imaging data.
 14. The method of claim12, wherein the one or more mechanical damages are selected from thegroup consisting of tearing, shredding, blade marks, wrinkling,cracking, bubbles, insufficient tissue sample, incomplete tissue sample.15. A method for quality control in histology system comprising:receiving a tissue block comprising a tissue sample embedded in anembedding material; imaging the tissue block to create a first imagingdata of the tissue sample in a tissue section on the tissue block;removing the tissue section from the tissue block, the tissue sectioncomprising a part of the tissue sample; imaging the tissue section tocreate a second imaging data of the tissue sample in the tissue section;comparing the first imaging data to the second imaging data to confirmcorrespondence in the tissue sample in the first imaging data and thesecond imaging data based on one or more quality control parameters;identifying as non-confirming a tissue section if one or more mechanicaldamages are present in the tissue sample in the second imaging data butnot in the first imaging data; and adjusting one or more operatingparameters associated with removing of the tissue section to correct oneor more mechanical damages, wherein the tissue section is non-confirmingif there is no correspondence in one or more quality control parametersin the tissue sample in the first imaging data and the second imagingdata, wherein the one or more quality control parameters include one ormore of shape of the tissue sample, size of the tissue sample, or one ormore mechanical damages, and wherein the one or more mechanical damagesare selected from the group consisting of tearing, shredding, blademarks, wrinkling, cracking, bubbles, insufficient tissue sample,incomplete tissue sample.
 16. A method for quality control in histologysystem comprising: receiving a tissue block comprising a tissue sampleembedded in an embedding material; imaging the tissue block to create afirst imaging data of the tissue sample in a tissue section on thetissue block; removing the tissue section from the tissue block, thetissue section comprising a part of the tissue sample; imaging thetissue section to create a second imaging data of the tissue sample inthe tissue section; comparing the first imaging data to the secondimaging data to confirm correspondence in the tissue sample in the firstimaging data and the second imaging data based on one or more qualitycontrol parameters, identifying as non-confirming a tissue section ifone or more mechanical damages are present in the tissue sample in thesecond imaging data but not in the first imaging data; and approving thetissue section if there are no mechanical damages are present in thetissue sample in the first imaging data and the second imaging data,wherein the tissue section is non-confirming if there is nocorrespondence in one or more quality control parameters in the tissuesample in the first imaging data and the second imaging data, whereinthe one or more quality control parameters include one or more of shapeof the tissue sample, size of the tissue sample, or one or moremechanical damages, wherein the one or more mechanical damages areselected from the group consisting of tearing, shredding, blade marks,wrinkling, cracking, bubbles, insufficient tissue sample, incompletetissue sample.
 17. A method for quality control in histology systemcomprising: receiving a tissue block comprising a tissue sample embeddedin an embedding material; imaging the tissue block to create a firstimaging data of the tissue sample in a tissue section on the tissueblock; removing the tissue section from the tissue block, the tissuesection comprising a part of the tissue sample; imaging the tissuesection to create a second imaging data of the tissue sample in thetissue section; comparing the first imaging data to the second imagingdata to confirm correspondence in the tissue sample in the first imagingdata and the second imaging data based on one or more quality controlparameters, identifying as non-confirming a tissue section if one ormore mechanical damages are present in the tissue sample in the secondimaging data but not in the first imaging data; and rejecting the tissueblock if one or more mechanical damages are present in both the firstimaging data and the second imaging data, wherein the tissue section isnon-confirming if there is no correspondence in one or more qualitycontrol parameters in the tissue sample in the first imaging data andthe second imaging data, wherein the one or more quality controlparameters include one or more of shape of the tissue sample, size ofthe tissue sample, or one or more mechanical damages, and wherein theone or more mechanical damages are selected from the group consisting oftearing, shredding, blade marks, wrinkling, cracking, bubbles,insufficient tissue sample, incomplete tissue sample.
 18. A method forquality control in histology system comprising: receiving a tissue blockcomprising a tissue sample embedded in an embedding material; imagingthe tissue block to create a first imaging data of the tissue sample ina tissue section on the tissue block; removing the tissue section fromthe tissue block, the tissue section comprising a part of the tissuesample; imaging the tissue section to create a second imaging data ofthe tissue sample in the tissue section; and comparing the first imagingdata to the second imaging data to confirm correspondence in the tissuesample in the first imaging data and the second imaging data based onone or more quality control parameters, wherein one or both of theimaging steps comprise: imaging a tissue section at one or morewavelength ranges; creating an imaging data of the tissue section;segmenting the tissue sample from the embedding material based on acolor and intensity information in the color imaging data; andidentifying a size, a shape or edges of the tissue sample in the tissuesection.
 19. A method for quality control in histology systemcomprising: receiving a tissue block comprising a tissue sample embeddedin an embedding material; illuminating the tissue block with UV light;imaging the tissue block, prior to removing one or more tissue sections,to generate a baseline imaging data of the tissue sample; imaging thetissue block to create a first imaging data of the tissue sample in atissue section on the tissue block; removing the tissue section from thetissue block, the tissue section comprising a part of the tissue sample;imaging the tissue section to create a second imaging data of the tissuesample in the tissue section; and comparing the first imaging data tothe second imaging data to confirm correspondence in the tissue samplein the first imaging data and the second imaging data based on one ormore quality control parameter.
 20. A method for quality control inhistology system comprising: receiving a tissue block comprising atissue sample embedded in an embedding material; imaging the tissueblock, prior to removing one or more tissue sections, to generate abaseline imaging data of the tissue sample; imaging the tissue block tocreate a first imaging data of the tissue sample in a tissue section onthe tissue block; removing the tissue section from the tissue block, thetissue section comprising a part of the tissue sample; imaging thetissue section to create a second imaging data of the tissue sample inthe tissue section; comparing the first imaging data to the secondimaging data to confirm correspondence in the tissue sample in the firstimaging data and the second imaging data based on one or more qualitycontrol parameter; and comparing an outline, size, or shape of thetissue sample in the first imaging data, the second imaging data or bothto an outline, size, or shape of the tissue sample expected from thebaseline imaging data.